The Afghan president has ordered US special forces to leave Wardak province within two weeks.
The decision was being taken due to allegations of disappearances and torture by Afghans considered to be part of US special forces, said a spokesman for Hamid Karzai.
The strategically significant, central province of Wardak has been the recent focus of counter-insurgency operations.
A US statement said it took all allegations of misconduct seriously.
But the US could not comment specifically on this latest development "until we have had a chance to speak with senior government officials", the statement by a spokesman for US special forces said.
"This is an important issue that we must discuss with our Afghan counterparts," the statement said.
The Afghan president's office said the decision to order the expulsion of US special forces had been taken at a meeting of the National Security Council.
"After a thorough discussion, it became clear that armed individuals named as US special force[s] stationed in Wardak province engage in harassing, annoying, torturing and even murdering innocent people," it said.
"A recent example in the province is an incident in which nine people were disappeared in an operation by this suspicious force and in a separate incident a student was taken away at night from his home, whose tortured body with throat cut was found two days later under a bridge.
"However, Americans reject having conducted any such operation and any involvement of their special force.
"The meeting strongly noted that such actions have caused local public resentment and hatred."
The presidential statement said Afghan forces were "duty bound" to put a stop to such behaviour, and urged local people to co-operate in bringing them to justice.
In a hastily convened news conference, a presidential spokesman suggested many of the allegations centred on Afghan citizens he alleged were working with US special forces.
"There are some individuals, some Afghans, who are working within these cells, within these [US] special forces groups" in Wardak province, said spokesman Aimal Faizi.
"But they are part of US special forces according to our sources and according to our local officials working in the province," he said.
He said all special forces must leave Wardak within two weeks.
All operations by international special forces in the province have also been ordered to stop with immediate effect.
Wardak is seen as a gateway for the Taliban to target Kabul, says the BBC's Karen Allen in the capital.
She says this move to expel US forces has come as something of surprise for the Americans.
There is not much clarity as to who these Afghans are, our correspondent says - not, it seems, the local police who have come in for criticism in the past.
The accountability of US forces and local militia working with them has been a growing source of friction in Afghan-US relations.
A week ago, Mr Karzai banned Afghan forces from calling in foreign air strikes on residential areas, following the deaths of 10 civilians in a night raid in eastern Kunar province.
Mr Karzai gave a blunt statement for the reasons for the ban.
"Our forces ask for air support from foreigners and children get killed in an air strike," he said.
The argument over accountability comes against a backdrop of long-term negotiations over which foreign forces will remain in Afghanistan after Nato's exit in 2014.
The bulk of Nato's 100,000 troops are due to leave by the end of that year.