Two Florida healthcare workers who came in contact with a confirmed Mers case are being tested for the virus after beginning to show flu-like symptoms.
Officials at Dr P Phillips Hospital said one worker is in hospital and the second is being isolated in his home.
On Monday, the second US case of Middle East respiratory syndrome (Mers), which has killed at least 145 people elsewhere, was confirmed.
President Barack Obama has been briefed by his advisers on the two US cases.
White House spokesman Jay Carney said: "The president has been briefed on this development. The CDC [US Centers for Disease Control and Prevention] is taking the current situation very seriously and is working in close coordination with local health authorities."
The "imported cases" had been both healthcare workers from Saudi Arabia, the country where most of the deaths have occurred.
Health officials say Mers only appears to spread through close contact, but there is no known cure.
Risk to US 'low'
Hospital officials in Orlando told reporters the second confirmed US case had made a visit to the Orlando Regional Medical Center last week, but only to accompany another person having a medical procedure.
The patient, a healthcare worker who lived in Saudi Arabia, had symptoms at the time, but did not seek treatment.
He was later admitted to hospital on 8 May. He is said to be doing well and is isolated in hospital.
Five healthcare workers from the regional medical centre and another 15 from the Dr P Phillips Hospital are being tested for Mers, including the two who have developed symptoms.
Hospital officials said they are awaiting test results to confirm any secondary infections.
The CDC has said the overall risk to the US is "very low" and says it does not appear the virus has changed.
Mers causes coughing, fever and sometimes fatal pneumonia, and is a virus from the same family as Sars, severe acute respiratory syndrome, which killed around 800 people worldwide.
The World Health Organisation (WHO) has reported a total of 536 cases of Mers since 2012, the majority inside Saudi Arabia, and 145 deaths.