Pakistan's government "sanctioned" the killing of journalist Saleem Shahzad, the top officer in the US military, Admiral Mike Mullen, has said.
But he said he could not confirm if the country's powerful intelligence agency, the ISI, was involved.
The Pakistani government called the statement "extremely irresponsible". The ISI has denied any involvement.
Adm Mullen's remarks are the most explicit to date in a downward spiral in recent US-Pakistan relations.
Mr Shahzad was kidnapped near his home in Islamabad in May. His body was found two days later in Punjab province. At the time, many in the Pakistani media blamed the Pakistan's intelligence agency for the murder, despite its denial.
An inquiry set up by the government to investigate the killing began work last month.
"I have not seen anything that would disabuse that report that the government knew about this," Adm Mullen told journalists in Washington on Thursday.
"It was sanctioned by the government, yeah," he said.
Adm Mullen added that he did not have a "string of evidence" linking the death to the ISI.
Pakistan had set up an independent commission into the killing and Adm Mullen's statement would not help that investigation, a government spokesman said. This point was repeated by Husain Haqqani, Pakistan's ambassador to the US.
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M Ilyas Khan
BBC News, Islamabad
Admiral Mullen's latest salvo is being interpreted here as an attempt by the US to keep pressure on the Pakistani security establishment at a time when the latter is engaged in a vigorous PR campaign to project a softer image of the military.
US-Pakistan relations visibly deteriorated with the Raymond Davis affair in March 2011 - when a CIA agent shot and killed two Pakistani men. Two months later US special forces killed Osama Bin Laden on Pakistani territory.
In subsequent weeks, there have been high profile visits by US officials to Pakistan, creating an impression that both sides are determined to move on from their mutual troubles.
But Mr Mullen's statement indicates that differences between the two countries not only persist, they are becoming serious enough to warrant public accusation by a high-ranking US official that Pakistani government "sanctioned" the murder of journalist Saleem Shahzad.
"Any evidence that our American friends have should be shared with that commission," Mr Haqqani told the New York Times newspaper.
"We are as interested in getting to the bottom of this matter as anyone else in the world, given our concern about human rights," he said.
Nevertheless, correspondents say, the remarks will further worsen ties between Washington and its ally, Islamabad.
Tensions hit news lows after the killing of al-Qaeda leader Osama Bin Laden by US commandos in the Pakistani city of Abbottabad.
Last month Adm Mullen publicly acknowledged that there would be substantial cuts to US military numbers in Pakistan.
He said that US-Pakistan ties needed time to heal but added that it would be dangerous to abandon Islamabad.
"I think the worst thing we could do would be cut them off," he said. If that happened, he said, "10 years from now, 20 years from now, we go back and it's much more intense and it's much more dangerous," he said.
Correspondents say that during his tenure Adm Mullen has been a forceful advocate for maintaining dialogue with Pakistan and with its military establishment.
He was said to be close to the Pakistani army's chief of staff, Gen Ashfaq Kayani. Indeed Adm Mullen is thought to have made more visits to Pakistan than any other senior US official or chief of staff in recent times. But, correspondents say, the latest comments are yet more evidence of his patience wearing thin.
He steps down as chairman of the Joint Chiefs of Staff later this year.
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KARACHI MIILITANT ATTACK
On 22 May a deadly 15-hour siege on Pakistan's Mehran naval airbase in Karachi left 16 dead
On May 27 Saleem Shahzad published a piece about al-Qaeda infiltration in Pakistan's navy
A number of journalists have speculated that the attackers may have had inside help
An ex-naval commando was arrested in connection wih the attacks on 30 May
'New kind of militant' behind Pakistan Karachi attack
Mr Shahzad, who worked for the Hong Kong-based Asia Times Online and the Italian news agency Adnkronos International, had made a career writing about various Islamist militant networks operating in Pakistan.
Shortly before his death he had written an article about al-Qaeda infiltration in Pakistan's navy.
He reported that the militant group was behind a recent deadly assault on the Mehran base in Karachi because talks had failed over the release of several naval personnel arrested on suspicion of links to al-Qaeda affiliates.
Human rights groups have called Pakistan the most dangerous place in the world for journalists to operate, saying they were under threat from Islamist militants but also Pakistan's military and intelligence agencies.