Rear Admiral John Kirby said the US had “reason to suspect that friendly fire is the cause here, specifically friendly fire from the air”.
He said the Pentagon “would let investigators do their work”.
Afghan officials say coalition forces had called for air support to fend off a Taliban attack in Zabul province.
An Afghan soldier and an interpreter were killed in the incident.
On Tuesday, White House press secretary Josh Earnest said President Barack Obama had been informed of the deaths and that his thoughts and prayers were with the families of those killed.
The incident is among the most serious cases of so-called “friendly fire” in Afghanistan, US military sources confirmed on Tuesday.
NATO-led troops have been battling Taliban and other insurgents in the country since 2001. Militants have stepped up attacks as foreign combat troops leave this year.
US defense officials told the Associated Press news agency the Americans killed were special operations forces.
Those elite troops are responsible for calling in air support. Under constraints imposed by President Hamid Karzai, they may only do so when they fear they are about to be killed, after concerns over civilian deaths.
The ISAF force currently has soldiers from 50 contributing nations in Afghanistan. Most troops stationed in the south are American.
The incident happened in Arghandab district, a place hotly contested between the Taliban and international forces for some years.
There have been more than 30 NATO forces killed this year in Afghanistan – the latest incident is the deadliest so far in 2014.
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