The patrol was attacked near Deh Rawood in Uruzgan province, a military spokesman said, an area where the military has clashed repeatedly with Taliban militants.
"We're sorry to say that two U.S. soldiers were killed and one U.S. soldier was injured," Maj. Mark McCann said.
The wounded soldier was taken to a nearby American base and was listed in stable condition.
The military withheld the victims' names until their families could be informed, and gave no further details of the incident.
A total of 110 soldiers have died since the United States attacked Afghanistan's former ruling Taliban and their al Qaeda allies in late 2001, according to U.S. government figures.
Many have died in roadside bombings in a swath of southern and eastern Afghanistan where Taliban and al Qaeda holdouts continue to defy a U.S. force currently numbering about 18,000.
The military also reported poorly aimed rocket attacks Wednesday on two of its bases in which one Afghan civilian died and another was injured.
Elsewhere, gunmen fatally shot a local Afghan security chief and two government soldiers in a Taliban stronghold, police said.
Four rockets were directed at the U.S. base in Orgun, a town near the Pakistani border in southeastern Paktika province, but missed the target, a military statement said.
Shrapnel killed one Afghan and injured another. The wounded man was in stable condition at an American base.
Four more rockets were fired at the main U.S. base at Bagram, north of the capital, Kabul. All landed outside the perimeter and caused no injuries.
The security chief died Tuesday when gunmen riding a motorcycle opened fire on his pickup truck in Deh Rawood district of Uruzgan province, said Rozi Khan, the provincial police chief.
Nasim Khan, the security chief of Girishk district in neighboring Helmand province, and two militiamen riding with him were killed, Rozi Khan said.
He blamed the attack on Taliban rebels who are active in the region and often ambush Afghan forces. He said authorities across the province were on the lookout for the two attackers.