A pregnant Iraqi woman in labour and her cousin were shot dead by US forces as they rushed to hospital along a closed road, police and relatives say.
The car's driver said he saw no warnings to stop
US forces said their car "entered a clearly marked prohibited area near coalition troops" in Samarra city and failed to heed warnings to stop.
The driver, who was injured, said he had not seen or heard any warnings.
Earlier, the US military announced all troops in Iraq would be trained in moral and ethical conduct in combat.
It comes in the wake of allegations that US marines deliberately killed more than 20 civilians in the town of Haditha last November.
Meanwhile, Iraq's prime minister said he would name his choices for defence and interior ministers on Sunday.
Nouri Maliki said it had been impossible to reach agreement between the different parties on who should fill the posts, so he would put his own choices forward to parliament for a vote.
"Consensus over the names of the heads of interior and defence is impossible, that's why I will present the names directly to parliament," he said.
In the shooting incident, which happened on Tuesday but was only fully reported on Thursday, pregnant Nabiha Nisaif Jassim, 35, and her 57-year-old cousin Saliha Mohammed Hassan were killed.
The pregnant woman's brother, who was driving his sister to the maternity hospital, was wounded by broken glass.
Relatives of the victims are in mourning
"I was driving my car at full speed because I did not see any sign or warning from the Americans," Khalid Nisaif Jassim said.
"It was not until they shot the two bullets that killed my sister and cousin that I stopped.
"God take revenge on the Americans and those who brought them here. They have no regard for our lives."
He said attempts to save the baby's life failed.
Local police told AFP news agency: "They took a wrong road just behind the hospital which is now closed because it is next to a military road used by the Americans."
US forces said: "As the vehicle neared the troop location and failed to stop despite repeated visual and auditory signals, disabling shots were fired into the vehicle.
"The loss of life in these incidents is regrettable and coalition forces go to great lengths to avoid them," a statement said.
The army said it had received reports from Iraqi police that "one of the females may have been pregnant" and they were investigating.
There were reports of various deadly incidents across Iraq on Thursday.
A barrage of mortar bombs landed in the southern Doura district of Baghdad, killing nine people and wounding at least 40.
A bomb killed two Iraqis and wounded another 21, as they milled around hoping for construction work in a central Baghdad square, police said.