Pakistani authorities have previously denied involvement in or advanced knowledge of a drone strike the United States said it carried out in Kabul in July that killed al Qaeda leader Ayman al-Zawahiri.
Afghan Acting Minister of Defence Mullah Mohammad Yaqoob told a news conference in Kabul that American drones had been entering Afghanistan via Pakistan.
"According to our information the drones are entering through Pakistan to Afghanistan, they use Pakistan's airspace, we ask Pakistan, don't use your airspace against us," he said.
A spokesperson for the US Central Intelligence Agency declined to comment.
Pakistan's foreign minister Bilawal Bhutto-Zardari told Reuters he had made checks after the air strike and had been told that Pakistani airspace was not used. He said he would check again after Sunday's allegations but expected the position to be the same.
"I really don't believe that this is a time that I wish to get into a debate with anyone or to have accusations ... frankly, I'm focused on the flood relief efforts," Bhutto-Zardari said in an interview, referring to deadly floods in Pakistan that have left millions of people homeless.
"The Afghan regime has promised not only to its own people but to the international community, that they will not allow their soil to be used for terrorists," he said.
Pakistan's foreign ministry released a statement saying it noted Yaqoob's comments with "deep concern."
"In the absence of any evidence, as acknowledged by the Afghan Minister himself, such conjectural allegations are highly regrettable and defy the norms of responsible diplomatic conduct," the statement said.
US Secretary of State Antony Blinken has said the Taliban "grossly violated" a 2020 agreement on the withdrawal of US-led forces from Afghanistan by hosting and sheltering Zawahiri.
The Taliban said it is investigating the July air strike and that it has not found the al Qaeda leader's body.
Yaqoob's comments could exacerbate tension between Afghanistan and its neighbor at a time when the Afghan Taliban is mediating talks between Pakistan and a Pakistani Taliban militant group.
Afghanistan, which is undergoing an acute economic crisis, also relies heavily on trade with Pakistan.