Over the last 18 months, even as the government battled a lawsuit filed by the first scientist, Steven J. Hatfill, investigators built a case against the second one, Bruce E. Ivins, a highly respected microbiologist who had worked for many years to design a better anthrax vaccine.

Last weekend, after learning that federal prosecutors were preparing to indict him on murder charges, Dr. Ivins, a 62-year-old father of two, took an overdose of Tylenol with codeine. He died in a Frederick hospital on Tuesday, leaving behind a grieving family and uncertainty about whether the anthrax mystery had finally been solved.

The apparent suicide of Dr. Ivins, a Red Cross volunteer and amateur juggler who had won the Defense Department’s highest civilian award in 2003, was a dramatic turn in one of the largest criminal investigations in the nation’s history. The attack, the only major act of bioterrorism on American soil, came in the jittery aftermath of the Sept. 11 attacks. It killed five people, sickened 17 others and set off a wave of panic.

In the early days after the letter attacks, in September and October 2001, Dr. Ivins joined about 90 of his colleagues at the Army Medical Research Institute of Infectious Diseases in a round-the-clock laboratory push to test thousands of samples of suspect powder to see if they were anthrax. Later, in April 2002, he came under scrutiny in an Army investigation of a leak of potentially deadly anthrax spores outside a sealed-off lab at Fort Detrick. He later admitted he had discovered the leak but not reported it.

Whether the focus on Dr. Ivins had resolved the case of the anthrax letters was unclear. A federal law enforcement official said that Dr. Ivins had been regarded as a strong suspect and that agents had been nearing an arrest, and a lawyer familiar with the investigation said he believed that prosecutors had planned to charge only Dr. Ivins. The link between Dr. Ivins’s suicide and the federal investigation was first reported on Friday in The Los Angeles Times.

But the Federal Bureau of Investigation declined on Friday to make public its case against Dr. Ivins, noting that evidence was under court seal as part of a grand jury investigation. Officials said they were briefing the victims of the anthrax letters — those who recovered, as well as family members of those who died — and would need to go to court to have evidence unsealed before it could even be summarized for the public.

A lawyer who had represented Dr. Ivins since May 2007, Paul F. Kemp, insisted that Dr. Ivins was innocent and had been driven to suicide by false suspicions.

“For six years, Dr. Ivins fully cooperated with that investigation, assisting the government in every way that was asked of him,” Mr. Kemp said in a written statement, calling the microbiologist “a world-renowned and highly decorated scientist who served his country for over 33 years with the Department of the Army.”

“We assert his innocence in these killings and would have established that at trial,” Mr. Kemp said. “The relentless pressure of accusation and innuendo takes its toll in different ways on different people, as has already been seen in this investigation.”

Mr. Kemp was clearly referring to the case of Dr. Hatfill, who was the focus of intensive F.B.I. and news media attention in the case beginning in mid-2002 and received a $4.6 million settlement from the government in June to settle a lawsuit accusing the F.B.I. and the Justice Department of destroying his career and personal life with leaks.

Whatever the cause of his suicide, Dr. Ivins had been behaving bizarrely in the weeks before his death. He was hospitalized briefly for depression and, according to a complaint filed with the police, threatened to kill a social worker who had treated him in group therapy, among others, in rants referring to his expectation that he would be charged with five counts of capital murder.

“It’s out of character,” said Norman M. Covert, a former spokesman and historian for the Army biodefense center who served with Dr. Ivins on an animal care committee. “But if the F.B.I. was really leaning on him, what a tremendous load that was on him.”

A spokesman for the Frederick police, Lt. Clark Pennington, said he could not say whether Dr. Ivins had left a suicide note because the anthrax investigation remained open.

Investigators in the huge inquiry traveled to many countries and by late 2006 had conducted 9,100 interviews, sent out 6,000 grand jury subpoenas and conducted 67 searches, the F.B.I. said. But the prime focus steadily narrowed: first to the Army infectious diseases laboratories, apparently linked to the letters by genetic analysis, then to Dr. Hatfill, a medical doctor who had become a bioterrorism consultant, and finally to Dr. Ivins, who worked in the same building as Dr. Hatfill and lived two blocks away from him outside the gates to Fort Detrick.

Two puzzles have haunted investigators from the beginning: the motive of the perpetrator and his skills. Because the notes in some of the letters mailed to news media organizations and two senators included radical Islamist rhetoric, investigators initially believed the letters might have been sent by Al Qaeda.

But the F.B.I. quickly settled on a different profile: a disgruntled American scientist or technician, perhaps one specializing in biodefense, who wanted to raise an alarm about the bioterrorism threat. [GR says: PHILLIP ZACK?] That theory accounted for the letters’ taped seams and the notes’ use of the word anthrax, a warning that allowed antibiotic treatment — not to be expected from a Qaeda attack intended mainly to kill.

That theory of a biodefense insider placed many scientists at the infectious diseases institute and other laboratories under scrutiny, even as they helped the F.B.I. analyze the anthrax powder in the letters.

“The F.B.I. would be remiss not to look at us, especially those of us who worked with anthrax,” said John W. Ezzell, an anthrax researcher who hired Dr. Ivins at the institute and knew him well. “We were all subjected to lie detector tests. We were all interviewed.”

Mr. Ezzell called Dr. Ivins “intense about his work, but a popular guy.” Asked whether he was aware that Dr. Ivins had become a more serious suspect, Mr. Ezzell declined to comment.

The other puzzle involved the skills necessary to produce the high-quality aerosol powder contained in the letters addressed to the senators, Tom Daschle, Democrat of South Dakota, and Patrick J. Leahy, Democrat of Vermont.

Scientists familiar with germ warfare said there was no evidence that Dr. Ivins, though a vaccine expert with easy access to the most dangerous forms of anthrax, had the skills to turn the pathogen into an inhalable powder.

“This is aerosol physics, not biology,” Dr. Zelicoff added. “There are very few people who have their feet in both camps.”

Mr. Ezzell said Dr. Ivins had worked on many projects involving anthrax spores and the toxin they produce, including experiments in which animals were exposed to anthrax to test vaccines. But he said the experiments, to his knowledge, involved anthrax spores in liquid and not in the dry powder form used in the letter attacks.

By their own admission, the F.B.I. and the Postal Inspection Service had little expertise in biological weapons in 2001, when they first loosed hundreds of agents on the investigation. Since then, at least 19 government and university laboratories have worked on the investigation, using clues like the genetic fingerprints of the anthrax, and radioactive isotopes in the water used to grow it, to try to trace it to a source.

The source, several officials said, was the infectious diseases institute, where the trail led to just a handful of vials in a single lab.

But the scientific evidence, some of it found using new methods, now may never be tested in a criminal trial, leaving questions about just how compelling it is.

“I would urge the bureau to publish its evidence if it declares the case solved and closed,” said Dr. Claire Fraser-Liggett, the former director of the Institute for Genomic Research, where the anthrax genome was decoded.

On Capitol Hill, where anthrax contamination in 2001 led to the evacuation of many offices, several members of Congress voiced skepticism about reports that the hunt for the anthrax killer might be over.

Representative Rush Holt, a Democrat whose district includes the Princeton, N.J., mailbox where investigators believe the letters were mailed, said the F.B.I. should provide a full briefing.