The US military's top officer, Adm Mike Mullen, has accused Pakistan's spy agency of having links with militants targeting troops in Afghanistan.
He said Pakistan's Inter-Services Intelligence (ISI) had a "long-standing relationship" with a militant group run by Afghan insurgent Jalaluddin Haqqani.
The comments came as he held talks in Islamabad on Wednesday. Pakistani officials are also in the US for talks.
Pakistan routinely rejects charges of collusion with militants.
The BBC's M Ilyas Khan in Islamabad says that US officials have in the past spoken anonymously or in circumspect terms about associations between the Pakistani establishment and insurgents.
But that with this blunt statement Adm Mullen has for the first time claimed a clear link between the two, our correspondent says.
"It's fairly well known that the ISI has a long-standing relationship with the Haqqani network," Adm Mullen told Pakistan's Dawn newspaper.
"Haqqani is supporting, funding, training fighters that are killing Americans and killing coalition partners. And I have a sacred obligation to do all I can to make sure that doesn't happen."
He said the spy agency's support of the network remained at the "core ... and the most difficult part of the relationship" and that he would take it up with Pakistan's army chief Gen Ashfaq Kayani.
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