The AC-130 gunship killed the five men Saturday night as they left a compound where Taliban leaders gathered, said military spokesman Lt. Col. Bryan Hilferty.
"Our aircraft did not engage noncombatants," Hilferty said. "We clearly identified and engaged five armed adult males in the open."
Afghan officials assert the airstrike killed four men, four children and three women.
People from the Char Chino district, about 250 miles southwest of the capital, met with the governor of Uruzgan province to protest the deaths, said local official Abdul Rahman.
Hilferty said U.S. forces called in the airstrike when armed men moved toward special forces troops surrounding the compound to arrest suspected members of the hard-line Taliban movement that ruled Afghanistan until being ousted by a U.S.-led coalition in 2001.
Crowds of armed men emerged from nearby compounds after the airstrike and the American forces, accompanied by Afghan troops, withdrew to avoid a major battle.
U.S. officials vowed to review the procedures for airstrikes after killing 15 Afghan children in two raids last month, drawing strong protests from Afghan officials and the United Nations.
Rahman insisted those killed Saturday were civilians.
"They were simple villagers. They were not Taliban," he said. "I don't know why the U.S. bombed this home."
Rahman complained that coalition forces hunting for fugitives were failing to communicate with local officials, who could capture and suspected militants if asked by the U.S. military.
The alleged bombing this week, and the deadly raids last month, are not the first incidents in which U.S. troops have killed civilians. Last April, an American warplane mistakenly bombed a house, killing 11 civilians near Afghanistan's eastern border with Pakistan, the U.S. military said.
In July 2002, more than 40 Afghan civilians were killed and more than 100 others were injured when a U.S. AC-130 gunship fired on several villages in Uruzgan province. Among the dead were 25 people at a wedding celebration, Afghans said.
But Americans are also in the firing line.
A U.S. military spokesman said three U.S. soldiers were wounded when insurgents launched a surprise daylight raid on an American base in southern Afghanistan.
The soldiers were hit by shrapnel when about 15 attackers opened fire on the base at Deh Rawood in Uruzgan province early Sunday with rocket-propelled grenades, machine guns and AK-47 assault rifles, Lt. Col. Bryan Hilferty said.
One attacker was killed when American soldiers returned fire.
The injured soldiers, who were flown to the main U.S. military base at Bagram, 250 miles northeast near the capital, Kabul, were in stable condition "and are expected to recover," Hilferty said.
It was unclear whether the attackers were affiliated with the Taliban or other groups who have mounted a wave of assaults on soldiers, government targets and aid workers across the south and east.
At least 45 people, most of them Afghan civilians, have died in violence since the passing of the country's first post-Taliban constitution on Jan. 4.
American bases have also come under attack, usually from misdirected rockets launched under cover of darkness, and Sunday's raid was unusually bold.
Hilferty said Afghan authorities and the 11,000-strong U.S.-led coalition force had control all over the country, but couldn't prevent insurgents from mounting "localized" attacks.
He dismissed Sunday's direct daylight raid as "a sign of desperation. It's not typically what they do. If they attack us like that, we'll kill 'em."
Meanwhile, President Hamid Karzai ordered the release Sunday of more former Taliban fighters, a highly symbolic move that could boost his standing and undermine holdouts from the ousted hard-line Islamic regime.