Pakistani security officials accused NATO of killing at least 24 soldiers in helicopter attacks on two border posts Saturday, while unofficial sources placed the death toll higher, dpa reported.
In protest, Pakistani authorities stopped NATO supplies from reaching landlocked Afghanistan.
The pre-dawn attack occurred in Mohmand Agency, one of the seven Pakistani tribal districts from where Taliban and al-Qaeda fighters infiltrate into Afghanistan to target NATO-led forces.
A military spokesman said NATO helicopters had targeted the two border posts without any provocation.
Twenty-four soldiers were "martyred" and 13 others were injured, the spokesman in a statement, which also carried a condemnation of the attack by the Pakistan Army chief.
However, one official at the paramilitary Frontier Corps headquarters in Peshawar, the capital of north-western Khyber-Pakhtunkhwa province, said that two officers had been killed in addition to the 24 confirmed deaths, and that 23 troops had been wounded.
"Pakistani troops effectively responded immediately in self-defence to NATO/ISAF's aggression with all available weapons," the Pakistani military said in a statement.
The military chief, General Ashfaq Parvez Kayani, lauded Pakistan's reaction.
US Ambassador Cameron Munter was summoned, on instructions from Prime Minister Yousuf Raza Gilani, to lodge a strong protest on the unprovoked NATO/ISAF attack, according to a foreign ministry statement.
"Strong protests have also been lodged in Washington and at the NATO Headquarters in Brussels," the statement read.
The International Security Assistance Force (ISAF) in Afghanistan promised a thorough investigation.
"This incident has my highest personal attention and my commitment to thoroughly investigate it to determine the facts," said ISAF commander General John R. Allen.
"My most sincere and personal heartfelt condolences go out to the families and loved ones of any members of Pakistan Security Forces who may have been killed or injured," he added.
The incident was expected to further strain relations between Pakistan and the United States, coming in the aftermath of the controversial killing of al-Qaeda chief Osama bin Laden by US special forces in Pakistan's north-western city of Abbottabad on May 2.
Gilani cut short a tour to home town in the central province of Punjab and convened a meeting of the Defense committee.
The airstrike triggered a wave of anger in the country, where anti-American sentiments already run high. Condemnation also came from opposition political parties.
"This is an attack on Pakistan's sovereignty and such attacks will not be further tolerated," said provincial governor Masood Kausar, who also heads the civilian government in seven tribal districts.
The border posts in Salala area are situated on hills along a route frequently used by Islamist militants to enter into Afghanistan, said an intelligence official who also spoke on condition of anonymity.
Jamil Khan, a senior security official in Khyber Agency, said trucks carrying supplies for NATO in Afghanistan, including food, fuel and weapons, had been stopped from crossing the border.
"So far, 150 trucks have been turned back," he said.
Pakistani officials said a second supply route running from the southern port city of Karachi to the border crossing of Chaman, in south-western province of Baluchistan, had also been closed.