(CNN) -- NATO helicopters crossed into Pakistani airspace from Afghanistan in pursuit of insurgents over the weekend, killing 49 people, a spokesman told CNN Monday.
Crossing the border did not violate the International Security Assistance Force rules of engagement, Maj. Michael Johnson said.
Pakistan is very sensitive about United States-led military operations on its territory and issued a strong protest Monday.
Pakistan called the incursions "a clear violation and breach" of the United Nations rules for foreign forces in Afghanistan.
The United Nations "mandate terminates/finishes at the Afghanistan border. There are no agreed hot pursuit rules," Pakistan's Foreign Ministry said in a statement.
ISAF spokesperson Maj. Sunset Belinsky confirmed later Monday that U.S. helicopters were involved in at least three border incidents over the weekend.
According to Belinsky, the first one on Saturday was "self-defense," with Apache helicopters pursuing insurgents across the border about a kilometer (about a half a mile) into Pakistani territory and opening fire.
Belinsky said the NATO forces communicated with the Pakistani military after the incident, in which the insurgents had opened fire on the helicopters and then crossed the border into Pakistan.
After that, a second NATO weapons team went to assess what happened in the first incident and came under fire from the Pakistani side of the border, Belinsky said. The second team returned fire, she said, adding it was unclear if the second team actually entered Pakistani air space.
Earlier, Johnson could not say whether the insurgents were killed in Afghanistan or Pakistan, or even whether the NATO helicopters fired their weapons in Pakistani territory.
The U.S. helicopeters were responding to an insurgent attack on a remote Afghan National Security Force outpost in Khost province on Friday, ISAF said in a statement.
"An air weapons team in the area observed the enemy fire, and following International Security Assistance Force rules of engagement, crossed into the area of enemy fire," the statement said.
Johnson, the ISAF spokesman, could not say how often NATO forces crossed into Pakistani airspace -- but said it had happened again Monday morning.
NATO is still getting the details on that incident, which he called "much smaller."
CNN's Richard Allen Greene, Fred Pleitgen and Nicola Hughes contributed to this report.