The tape apparently was made before the announcement that Iraq's most-wanted terrorist, Abu Musab al-Zarqawi, was killed Wednesday by a U.S. airstrike. Al-Zawahiri praised the Al Qaeda in Iraq leader's efforts to confront U.S.-led forces in Iraq.
"God bless the prophet of Islam in Iraq, the persistent hero of Islam, the Holy Warrior Abu Musab al-Zarqawi," al-Zawahiri said.
He also sent greetings to the Shura Council of Mujahedeen in Iraq and insurgents, "who are confronting crusaders and their apostate aides and the merchants of religion."
The bearded al-Zawahiri wore a white turban and tunic, and sat before a black background with an automatic weapon propped behind his right shoulder. He repeatedly waved his right hand and pointed his finger in a gesture of admonishment.
Al-Zawahiri urged Muslims to reject a referendum proposed by Palestinian President Mahmoud Abbas on establishing a Palestinian state alongside Israel.
"I call on Muslims to reject any referendum on Palestine, because Palestine is a house of Islam and not subject to any compromise," he said.
"I call upon Muslims everywhere to support the brothers in Palestine," he said, mentioning armed Islamist militants, prisoners and their families.
Al-Zawahiri, who has focused on political issues before in his tapes, also discussed the campaign by a group of Egyptian judges for the independence of the judiciary and the ongoing violence in Sudan's Darfur region.