The NATO-led force said a man wearing an Afghan army uniform opened fire on the Americans in Paktika province, close to the border with Pakistan.
"An individual wearing ANA (Afghan National Army) uniform turned his weapon against the ISAF service members," an ISAF statement said.
"Today was a very difficult day for ISAF," spokesman Brig. Gen. Guenter Katz said.
In a separate attack, an Italian soldier died in Farah province in the west.
The coalition said the soldier was killed "during an attack by an enemy of Afghanistan".
It was an "insurgent attack", rather than an insider incident, ISAF said.
The Italian defence ministry said the soldier was killed by "hostile elements" and three more were wounded.
A Lynx vehicle was attacked at 10.30 am as it was returning to base after training Afghan security forces, BBC said.
Reports said a grenade was thrown at the vehicle by a man wearing an Afghan army uniform.
The dead soldier was named as Giuseppe La Rosa, a 31-year-old member of the Third Bersaglieri Regiment.
If the attacker in Paktika is confirmed to be a member of the Afghan army, Saturday's incident will be the fifth insider attack in the country since the beginning of this year.
Four American and one British soldier and a civilian contractor of the ISAF have been killed in four insider attacks this year, Xinhua reported.
In 2012, more than 45 such incidents took place, killing 60 foreign soldiers across the country.
Saturday's deaths have brought to 85 the number of foreign soldiers killed in Afghanistan since January.