A US Congressional panel has frozen $700m (£450m) in aid to Pakistan until it gives assurances it is tackling the spread of homemade bombs in the region.
The move - the second such freeze this year - reflects US frustration over what it sees as Islamabad's reluctance to act against militant groups.
But it has has been criticised by senior Pakistani politicians.
The killing of Osama Bin Laden by US forces and ongoing US drone strikes in Pakistan have strained bilateral ties.
Washington is also known to be unhappy about what it sees as lacklustre Pakistani efforts to counter the al Qaeda-linked Haqqani network, which it believes operates out of Pakistan and fights US troops in Afghanistan.
Correspondents say that Pakistan is one of the largest recipients of American foreign aid and the cutback announced on Tuesday is only a small proportion of the billions of dollars it receives from Washington every year in civil and military assistance.
But the freeze in aid - part of a defence bill that is expected to be passed by Congress later this week - could presage even greater cuts, correspondents say.
Washington has provided about $20bn (£12.8bn) in security and economic aid to Pakistan since 2001, much of it in the form of reimbursements for assistance in fighting militants.
In July the US said it was withholding some $800m (£500m) in military aid to Pakistan - about a third of the annual US security assistance to Pakistan.
White House Chief of Staff Bill Daley told ABC television at that time that Pakistan had "taken some steps that have given us reason to pause on some of the aid".
Justifying the latest aid freeze, some in Congress say that Islamabad has not only failed to act against militant groups but that in some cases it has actively provided them with help, a charge Pakistani officials deny.
Members of Congress are particularly aggrieved over suspicions that homemade bombs - or improvised explosive devices (IEDs) - are being made by militants based in Pakistan for use against US and Nato troops in Afghanistan.
IEDs are among the most effective weapons of the militants, and are responsible for most coalition casualties in Afghanistan.
Many are reportedly made using ammonium nitrate, a common fertiliser which Washington believes is being smuggled across the border from Pakistan.
The US wants "assurances that Pakistan is countering improvised explosive devices in their country that are targeting our coalition forces", Representative Howard McKeon, a House Republican, said.
Pakistan, however, argues that it is doing its utmost to fight al-Qaeda and the Taliban - and hundreds of its soldiers have been killed since it joined the US-led war in Afghanistan in 2001.
"It [the latest freeze] is most unfortunate and untimely," Pakistani senate committee on foreign affairs chairman Salim Saifullah Khan told the AFP news agency.
"I think we will survive without aid, but it is most unfortunate to see these things after 31 years of sacrifices by Pakistan."
Last month Pakistan accused Nato of killing 24 Pakistani soldiers in an air strike near the Afghan border - and has stopped fuel being supplied from Pakistan to Nato forces in Afghanistan as a sign of its anger.