The US says it has been sharing information with Pakistan for several years about the illegal proliferation of nuclear weapons technology.
Khan's public confession shocked the nation
State Department spokesman Richard Boucher said the issue had been a long-standing concern for both countries.
On Monday, Pakistani President Pervez Musharraf said the US gave him evidence of the illegal deals of Pakistan's top nuclear scientist only last October.
Last week, Abdul Qadeer Khan confessed he had sold nuclear secrets abroad.
Dr Khan, regarded as the "father" of Pakistan's nuclear bomb, publicly admitted that he had supplied nuclear technology to North Korea, Iran and Libya through a black market.
He was pardoned by President Musharraf on condition he would co-operate fully with the ongoing inquiry.
Mr Khan insisted he had acted alone, but many experts are questioning how he was able to do this without the knowledge of the Pakistani authorities, the BBC's Jannat Jalil in Washington reports.
"We have discussed non-proliferation issues with Pakistan repeatedly over a long period of time, and it's been an issue of concern to us and to President Musharraf," Mr Boucher said in Washington.
He said American officials had from time to time given Pakistan what he described as "pieces of information" on the issue.
The spokesman stressed that "certainly our non-proliferation dialogue with Pakistan goes back much further than" last October - as claimed by President Musharraf.
But Mr Boucher said he could not give more details, because that might reveal where America was getting its information from.
In an interview with the New York Times published earlier on Tuesday, President Musharraf said he had suspected for at least three years that Dr Khan was sharing nuclear technology with other countries.
But he said that he needed proof and only got that with the help of Washington last October.
"If they knew it earlier, they should have told us," the paper quotes the president as saying.
"Maybe a lot of things would not have happened."