The blockade appeared to be a major escalation in tensions between Pakistan and the United States.
A permanent stoppage of supply trucks would place massive strains on the relationship between the two countries and hurt the Afghan war effort. Even a short halt is a reminder of the leverage Pakistan has over the United States at a crucial time in the 9-year-old war.
By late afternoon, a line of more than 150 NATO vehicles was waiting to cross the border into Afghanistan, officials said.
"We will have to see whether we are allies or enemies," Pakistani Interior Minister Rehman Malik said of the border incident, without mentioning the blockade.
NATO said it was investigating Pakistani reports that coalition aircraft had mistakenly attacked its forces. The coalition has on at least one other occasion acknowledged mistakenly killing Pakistani security forces stationed close to the border.
Over the weekend, NATO helicopters fired on targets in Pakistan at least two times, killing several suspected insurgents they had pursued over the border from Afghanistan. Pakistan's government protested the attacks, which came in a month during which there have been an unprecedented number of U.S. drone missile strikes in the northwest, inflaming already pervasive anti-American sentiment among Pakistanis.
Prime Minister Yousuf Raza Gilani told visiting CIA director Leon Panetta in Islamabad that Pakistan was "profoundly concerned" about the missile strikes and helicopter incursions. "Pakistan being a front-line ally in the war against terror expects its partners to respect its territorial sovereignty," he said, according to a statement from his office.
The surge in attacks and apparent increased willingness by NATO to attack targets on the border, or just inside Pakistan, could be a sign the coalition is losing patience with Pakistan, which has long been accused of harboring militants in its lawless tribal regions.
A Pakistani army statement said Thursday's airstrike targeted a checkpoint 650 feet (200 meters) inside Pakistan in the Upper Kurram tribal area, where six soldiers of the paramilitary Frontier Corps were stationed.
The Pakistani troops fired warning shots at the two helicopters, which responded with a pair of missiles that destroyed the post, killed three of the soldiers and wounded the other three, it said.
The statement was cautiously worded, saying the choppers "appeared to have crossed the border."
The bodies of the three dead men were taken to Parachinar, the region's largest town, one security official said.
Several hours later, officials reported another rocket strike by NATO helicopters about nine miles (15 kilometers) from the first one. There were no injuries.
The security officials spoke on condition of anonymity because of the sensitivity of the situation and because in some cases they were not authorized to release the information to the media.
The border between Pakistan and Afghanistan is unmarked. Border troops wear uniforms that resemble the traditional Pakistani dress of a long shirt and baggy trousers, which could make it hard to distinguish them from ordinary citizens or insurgents.
U.S. officials have complained in the past that Pakistani security forces do little to stop the movement of militants seeking to cross over into Afghanistan and attack foreign troops there.
Lt. Col. John Dorrian, a spokesman for intelligence and special operations at NATO headquarters in Kabul, said coalition forces observed early Thursday what they believed were insurgents firing mortars at a coalition base in Dand Wa Patan district of Paktia, which is next to Upper Kurram.
"A coalition air weapons team called for fire support and engaged the insurgents," he said. "The air weapons team reported that it did not cross into Pakistani air space and believed the insurgents were located on the Afghan side of the border."
Dorrian said Pakistani military officials had informed the NATO military coalition that members of their border forces had been struck by coalition aircraft. He said the coalition was reviewing the reports to see if the operation in Paktia was related to those reports.
Hours after the incident, Pakistani authorities were ordered to stop NATO supply trucks from crossing into Afghanistan at the Torkham border post, a major entryway for NATO materials at the edge of the Khyber tribal region, two government officials said.
No reason was given, but earlier this week Pakistan threatened to stop providing protection to NATO convoys if the alliance's helicopters attacked targets inside Pakistan again.
The other main route into Afghanistan in southeastern Pakistan had received no orders to stop NATO trucks from crossing, which they were doing as normal, said Syed Mohammed Agha, a spokesman for the Pashin Scouts border guards.
Pentagon officials said they were trying to clarify exactly what happened and were talking to the Pakistani government. A Defense Department spokesman said it was too soon to know what impact the border crossing closure would have.
"We expect this matter to be resolved through continued dialogue," Marine Corps Col. Dave Lapan said.
Some 80 percent of non-lethal supplies for foreign forces fighting in landlocked Afghanistan are transported over Pakistani soil after being unloaded at docks in Karachi, a port city in the south. While NATO and the United States have alternative supply routes, the Pakistani ones are the cheapest and most convenient.
In June 2008, a U.S. airstrike killed 11 Pakistani troops and frayed ties between the two nations. Pakistan said the soldiers died when U.S. aircraft bombed their border post in the Mohmand tribal region. U.S. officials said coalition aircraft dropped bombs during a clash with militants. They expressed regret over the deaths, but said the attack was justified.
Pakistan and the U.S. have a complicated, but vital, relationship, with distrust on both sides.
Polls show many Pakistanis regard the United States as an enemy, and conspiracy theories abound of U.S. troops wanting to attack Pakistan and take over its nuclear weapons. The Pakistani government has to balance its support for the U.S. war in Afghanistan - and its need for billions of dollars in American aid - with maintaining support from its own population.
Riechmann reported from Kabul, Afghanistan. Associated Press writers Munir Ahmed in Islamabad, Riaz Khan in Peshawar, Matiullah Achakzai in Chaman, and Pauline Jelinek in Washington, D.C. contributed to this report.