John Clifford, the US Department of Agriculture's (USDA) chief veterinary officer, confirmed a positive result for bovine spongiform encephalopathy (BSE) after two inconclusive test results over the weekend.
But he stressed that the animal, which aroused suspicions after losing the ability to walk, had been slaughtered and buried on the farm in Alabama without entering the food chain.
The USDA was working with Alabama veterinary officials to investigate the animal's herd of origin, Mr Clifford said in a statement.
"Experience worldwide has shown us that it is highly unusual to find BSE in more than one animal in a herd or in an affected animal's offspring. Nevertheless, all animals of interest will be tested for BSE," he said.
"I want to emphasise that human and animal health in the United States are protected by a system of interlocking safeguards, and that we remain very confident in the safety of US beef,” he said.
The US meat industry was hit by the news. Shares in hamburger chain McDonalds lost 33 cents to close at US$34.32, while meat-processing giant Tyson Foods fell 59 cents to US$13.06.
The few cases of BSE in the US pales compared to the more than 183,000 infections confirmed in Britain, where the epidemic was first identified in 1986.
The United States recorded its first case of BSE in December 2003 in a Canadian-born cow in Washington state. The second case was confirmed in June last year in a cow in Texas.
The discovery of BSE in 2003 prompted a slew of bans on US beef imports around the world, which have only recently been lifted in some countries.
Consumption of BSE-infected beef has been linked to a brain-wasting disease in humans called variant Creutzfeldt-Jakob disease (vCJD).
Mr Clifford said preliminary findings from Alabama indicated that the BSE-infected animal was likely to have been more than 10 years in age.
That would mean it was born before the US government banned the use of animal parts and bonemeal in cattle feed in 1997, to stop the most probable route for BSE transmission.
Most countries that have resumed US beef imports in recent months have specified they will only take beef from cattle aged 30 months or younger, in line with international scientific advice.
Japan, formerly the biggest overseas market for US beef, insisted on tougher guidelines so that only meat from cattle aged less than 20 months would be accepted.
But only a month after resuming the imports, Japan re-imposed its ban on US beef in January after a veal shipment from New York was found to contain forbidden spinal material.
Mr Clifford stressed "we will continue to be very transparent in sharing information with the public and with our trading partners around the world".
Jeremy Russell, spokesman for the National Meat Association, said the new case was "disappointing" but "not unexpected, given that this animal was an older one and was probably given animal feed before 1997".
Asked about the possible impact on US beef markets abroad, he told news agency AFP: "We would hope that there wouldn't be any, but given the past, it can't be ruled out. The science indicates there really shouldn't be any concern."