A US soldier and contractor were killed while three Afghan soldiers died and several were injured.
The new deaths occurred on Saturday in Wardak province.
The international mission, ISAF, initially said the soldier was believed to have been killed by a member of the Afghan security services, but it later said the circumstances were unclear.
What is known is that a firefight took place, after what ISAF described as a short conversation between coalition and Afghan soldiers.
ISAF says “insurgent fire” may have been involved in the incident, which is now under investigation by a joint Afghan and coalition team.
The American death toll goes back to the US-led invasion of Afghanistan in 2001.
Sunday’s incident took place at a checkpoint near an Afghan National Army base in the district of Sayedabad, according to Afghan officials.
Shahidullah Shahid, a provincial government spokesman, earlier told the Associated Press news agency that an Afghan soldier had turned his gun on Americans and started shooting.
“Initial reports indicate that a misunderstanding happened between Afghan army soldiers and American soldiers,” he said.
But ISAF later said an American soldier and an American contractor, along with three Afghan soldiers, were killed in an exchange of fire in confusing circumstances that may have involved insurgent activity.
ISAF’s deputy commander, Lt Gen Adrian Bradshaw, said: “The circumstances were somewhat confused and we are establishing the full facts to the extent that it is possible.”
Military officials from both sides have launched a joint investigation.
The figure of 2,000 deaths was given by US officials on Sunday. During the war in Iraq, 4,409 American soldiers were killed.
As of 27 September, the Pentagon’s official military death toll for Afghanistan had stood at 1,996.
The count includes both soldiers killed in action and soldiers who died of their injuries in hospital. The figure also covers 339 non-combat deaths.
A report by the Brookings Institution estimates that 40.2% of US deaths were caused by improvised explosive devices and 30.3% by gun attacks.
Officially, at least 17,644 US soldiers have been wounded in action in Afghanistan.
The independent organization iCasualties estimates a higher US death toll, recording 2,125 to date.
This same source reports 1,066 deaths of non-US members of the coalition in Afghanistan.
It is more difficult to establish the Afghan toll in the war but most estimates calculate a minimum of 20,000 civilian deaths, AP notes.
Some 10,000 members of the Afghan security forces have been killed. No reliable figures exist for deaths among the Taliban and other insurgents.
NATO combat troops are set to withdraw by the end of 2014, but a central plank of the strategy is that foreign soldiers will serve alongside and train Afghans for many years to come.
Correspondents say that may not be realistic given the ever increasing number of Afghans who turn their weapons on their foreign allies.
Afghan war deaths:
• 2,000 US soldiers
• 1,066 non-US coalition soldiers
• possibly 20,000 civilians
• 10,000 members of Afghan security forces
• hundreds of private contractors
• unknown number of insurgents
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