Pakistan has stopped US drone flights from a remote airbase in the western province of Balochistan and ordered US personnel to vacate it, the defence minister has said.
"We have told them to leave the Shamsi airbase," Chaudhry Ahmed Mukhtar said on Wednesday night, adding that US personnel had already started to shift equipment from the base.
A US embassy spokesman declined to comment, referring queries to Washington.
Shamsi is located in a remote valley 350 miles south-west of Waziristan, where most of the CIA-directed Predator and Reaper drone strikes against al-Qaida and Taliban targets take place.
The closure of the base is a blow to a covert programme that has killed up to 2,500 people since its inception seven years ago and forms a cornerstone of President Barack Obama's strategy to flush al-Qaida from its Pakistani havens.
The US insists it will press ahead with the strikes. In unusually direct comments, Obama's counter-terrorism adviser John Brennan said on Wednesday that the US would continue to "deliver precise and overwhelming force against al-Qaida" in the tribal areas.
The attacks are likely to continue from CIA bases in Afghanistan – the latest took place on June 20 in Kurram tribal agency. A senior Pakistani military official said the US had not used Shamsi for "several months" and was already flying drones across the border.
Senior civilian officials said they closed Shamsi in retaliation for an American reduction of coalition support funds, a multibillion-dollar subsidy for Pakistani military operations. The defence minister said US forces had already vacated Ghazi airbase, 40 miles north-west of Islamabad.
A US official in Pakistan accused the government of engaging in "diplomacy by headline" but refused to comment further.
The spat marks another low point in Pakistan-US relations after the raid to kill Osama bin Laden on 2 May and the furore over a CIA agent, Raymond Davis, who shot dead two men in Lahore in January.
Pakistan's military and the ISI intelligence service have sought to restrict CIA activities by seeking lists of spies, closing intelligence cooperation centres, and restricting visas for US personnel.
The US, meanwhile, is trying to repair the relationship, recognising Pakistan's importance in fighting al-Qaida and, perhaps, reaching a peace settlement in Afghanistan.
Although at least 120 military trainers have been ordered to leave the country, the US recently agreed to replace two Orion surveillance planes that were destroyed in a militant assault on a Karachi naval base in May.
The CIA use of Shamsi is controversial in Pakistan, where drone strikes are extremely unpopular. A recent Pew poll found 97% of respondents viewed them negatively.
Shamsi was built by Arab Sheikhs from the United Arab Emirates to facilitate hunting falcon trips for the houbara bustard, a rare bird some Arabs believe has aphrodisiac properties. The CIA presence was detected in 2004, when the first drone strikes occurred. Google Earth images showed Predator drones parked on the runway.
Since then CIA contractors have been stationed at Shamsi, fuelling and arming Predator and the newer Reaper drones. Operators at the base control the pilotless planes during takeoff but control quickly passes to a "reachback operator"sitting at a video screen thousands of miles away at the CIA headquarters in Langley Virginia.
The drones use different warheads, from Hellfire missiles that travel at supersonic speeds to laser-guided Stingers and other missiles using thermobaric warheads that create percussion waves which can penetrate deep bunkers and caves.
According to the New America Foundation, which tracks drone strikes, there have been 253 since 2004, with 42 so far this year. Various press reports put the death toll from the strikes at between 1,557 and 2,464.
The varying figures highlight the difficulty of obtaining accurate information from the tribal belt, which is out of bounds to foreigners and most local reporters, and where Taliban fighters take control of drone attack sites immediately after the strikes occur.
The most contentious issue is civilian casualties. The New American foundation, based on press reporters, estimates non-militant deaths at 20% of the total, although in 2010 this fell to 5%.
Pakistan's military has previously tried to distance itself from Shamsi by claiming that the airbase was the territory of the United Arab Emirates. However base security and other logistics have been provided by Pakistani forces.