"We will not negotiate with anyone over the undeniable right of nuclear technology and using it," said Ayatollah Khamenei.
On June 6th EU foreign policy chief Javier Solana presented Iran with an offer - backed by Britain, China, France, Germany, Russia and the United States - of multilateral talks and a variety of incentives.
The offer would pave the way for the first substantive contacts between Iran and the United States in decades, but it is conditional on Iran first agreeing to suspend enrichment work.
However, Iran appears to still reject the key condition and continues to call for negotiations without any "preconditions".
"If they recognise this right, we are ready to negotiate over supervision controls," Ayatollah Khamenei said of Iran's nuclear program, which the regime insists is merely aimed at generating electricity.
In Washington, the White House said it did not consider Ayatollah Khamenei's comment as the Islamic Republic's final word on the offer.
Spokesman Tony Snow said the United States would only recognise a formal answer given by Iran's senior nuclear negotiator, Ali Larijani, to European Union foreign policy chief Javier Solana.
The two are expected to meet in the coming days, although a precise time and venue have yet to be set.
"The position has always been the same, which is: When Mr Larijani communicates with Javier Solana that is how we expect to have an answer to the proposal," Mr Snow told reporters.
"There are differing voices coming out of Iran. This is the one that we and our EU partners have said would be the appropriate track for responding, and therefore that's the track that we would expect to hear through."
The US has insisted that Iran has "weeks, not months" to reply to the incentives package, but Iran appears to still reject the key condition and continues to call for negotiations without any "preconditions".