Rowhani's comments come ahead of a meeting in New York later in the month between his foreign minister Mohammad Javad Zarif and EU foreign policy chief Catherine Ashton on restarting negotiations over the Islamic republic's nuclear programme.
"Our government will not give up one iota of its absolute rights" on the nuclear issue, Rowhani said, repeating a mantra frequently used by his predecessor Mahmoud Ahmadinejad.
He also said that following the meeting between Zarif and Ashton, nuclear negotiations "will continue in another place with the 5+1 group," which is made up of the United States, Russia, China, France, Britain and Germany, but gave no further details.
On Friday, after a phone call with Ashton, the P5+1 lead negotiator, Zarif said that Tehran wanted to "remove any ambiguity" about its nuclear work.
Rowhani has also appointed a new Iranian envoy to the International Atomic Energy Agency, Reza Najafi, and former foreign minister Ali Akbar Salehi as head of Iran's Atomic Energy Organisation.
Salehi indicated last week that Iran could grant the IAEA greater inspection rights.
Rowhani said soon after his election as president in June that he wanted to hold "serious" talks "without wasting time" on Iran's nuclear file, while maintaining Iran's "undeniable rights" to its nuclear programme.
Ahmadinejad's refusal to make any concessions on the nuclear issue saw the Islamic republic slapped with rounds of international sanctions, which in particular targeted its oil exports and banking transactions.
Oil revenues have halved due to the sanctions, causing the value of the rial to plunge and inflation to soar to above 40 percent.
But Rowhani said that such measures would not make Iran abandon its programme.
"The West must understand that it will not obtain any result by threats and pressure," he said in his speech to the clerics.
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