The six glass vials were intact and sealed, and scientists have yet to establish whether the virus is dead or alive, the Centers for Disease Control and Prevention said Tuesday.
Still, the find was disturbing because for decades after smallpox was declared eradicated in the 1980s, world health authorities said the only known samples left were safely stored in super-secure laboratories in Atlanta and in Russia.
Officials said this is the first time in the U.S. that unaccounted-for smallpox has been discovered. But at least one leading scientist raised the possibility that there are more such vials out there around the world.
The CDC and the FBI are investigating.
It was the second recent incident in which a U.S. government health agency appeared to have mishandled a highly dangerous germ.
Last month, scores of CDC employees in Atlanta were feared exposed to anthrax because of a laboratory safety lapse. The CDC began giving them antibiotics as a precaution.
The freeze-dried smallpox samples were found in a building at the National Institutes of Health in Bethesda, Maryland, that has been used by the Food and Drug Administration since 1972, according to the CDC.
The scientist was cleaning out a cold room between two laboratories on July 1 when he made the discovery, FDA officials said.
Officials said labeling indicated the smallpox had been put in the vials in the 1950s. But they said it's not clear how long the vials had been in the building, which did not open until the 1960s.
No one has been infected, and no smallpox contamination was found in the building.
Smallpox can be deadly even after it is freeze-dried, but the virus usually has to be kept cold to remain alive and dangerous.
In an interview Tuesday, a CDC official said he believed the vials were stored for many years at room temperature, which would suggest the samples are dead. But FDA officials said later in the day that the smallpox was in cold storage for decades.
"We don't yet know if it's live and infectious," said Stephan Monroe, deputy director of the CDC center that handles highly dangerous infectious agents.
The samples were rushed to the CDC in Atlanta for testing, after which they will be destroyed.
Peter Marks, deputy director of the FDA's Center for Biologics Research and Evaluation, said the discovery was unexpected but not a total shock. He added, however, that "no one's denying we should have done a better job cleaning out what was there."
In at least one other such episode, vials of smallpox were found at the bottom of a freezer in an Eastern European country in the 1990s, according to Dr. David Heymann, a former World Health Organization official who is now a professor at the London School of Hygiene and Tropical Medicine.
Heymann said that when smallpox samples were gathered up for destruction decades ago, requests went out to ministers of health to collect all vials.
"As far as I know, there was never a confirmation they had checked in with all groups who could have had the virus," he said.
Dr. Donald "D.A." Henderson, who led the WHO smallpox-eradication effort and is now a professor at the Center for Health Security at the University of Pittsburgh, said it is highly unlikely more such stashes will be discovered. But he confessed that "things were pretty casual" in the 1950s.
Decades ago, he recalled, "I came back from many a trip carrying specimens, and I just put them in the refrigerator until I could get them to a laboratory. My wife didn't appreciate that."
Smallpox was one of the most lethal diseases in history. For centuries, it killed about one-third of the people it infected, and left most survivors with deep scars on their faces from the pus-filled lesions.
The last known case was in Britain in 1978, when a university photographer who worked above a lab handling smallpox died after being accidentally exposed to it through the ventilation system.
Global vaccination campaigns finally brought smallpox under control. After it was declared eradicated, all known remaining samples of live virus were stored at a CDC lab in Atlanta and at a Russian lab in Novosibirsk, Siberia.
The labs take extreme precautions. Scientists must undergo fingerprint or retinal scans to get inside, they wear full-body suits including gloves and goggles, and they shower with strong disinfectant before leaving the labs.
There has long been debate over whether to destroy the stockpile.
Many scientists argue that any remaining samples pose a threat and that the deadly virus should be wiped off the planet altogether. Others contend the samples are needed for research on better treatments and vaccines.
At its recent annual meeting in May, WHO put off a decision again.