Speaking late on Saturday, Mussa Abu Marzuq said Hamas, which recently signed a reconciliation deal with the Western-backed Palestinian leadership in the occupied West Bank, would never agree to recognise Israel.
"We will not recognise the Zionist entity," he said at a press conference in Gaza City.
Under terms of the deal, Gaza's Hamas rulers and the Palestine Liberation Organisation of president Mahmud Abbas are to work together to form a new unity government which will prepare for national elections.
Recognising Israel is one of the key conditions laid out in the 2003 peacemaking roadmap of the Middle East Quartet, which brings together the United Nations, the European Union, the United States and Russia.
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The other two key demands are a renunciation of violence and acceptance of all prior agreements with Israel.
Abbas, who is to head the new government, to consist of political independents, has insisted it will abide by all three principles.
"Hamas rejects the Quartet's conditions because it denies some of our people's rights," he told reporters.
"We will always refuse any conditions that deny our Palestinian rights."
He also said the question of disarming Hamas's armed wing, the Ezzedine al-Qassam Brigades, was "never mentioned" in talks with the PLO since the unity deal was inked on April 23. "No one asked to discuss this," he said.
Azzam al-Ahmad, a senior member of Abbas's Fatah movement, which dominates the PLO, was to arrive in Gaza City today or tomorrow to begin consultations on forming the new government, he said.