In Washington, a CIA spokeswoman said it was al-Zawahiri's voice on the tape.
A US counterterrorism official initially said the tape was several years old but then retracted the statement, saying that the date of the recording was uncertain. The website which posted the recording said it was recently recorded without giving a specific date.
"I am honored to recite this jihadist poem," al-Zawahiri said, praising mujahidin fighters. "We shall remain true to our oath (with the Afghan mujahidin)."
The message emerged one day after Bin Laden, al-Qaida's leader, said the group was preparing attacks in the United States but was open to a conditional truce with the Americans, according to an audiotape attributed to him.
In his 17-minute tape, al-Zawahiri praised an Islamist Afghan poet, Mohebullah Kandahari: "The owner of the sword and pen, who carried both a machine gun and the Quran, known in scholarly circles ... and who could be seen in jihadi arenas from the time of the Russian communist attack on Afghanistan until the crusader raid [led by the United States in 2001]."
The CIA spokeswoman said: "After conducting a technical analysis, the CIA concludes with high confidence that the voice on the tape is that of Ayman al-Zawahiri."
The little-known website which carried the tape said it was new, but the US counter-terrorism official cast doubt on that assertion. "There is no reason to believe it was done recently. It could have been done at any time," the official said.
The United States conducted an air attack in Pakistan last week, which officials said was aimed at al-Zawahiri and in which Pakistani intelligence sources said "a few militants" had been killed.
The last tape attributed to al-Zawahiri was issued earlier this month. He said US President George Bush's plans to withdraw troops from Iraq meant Washington had been defeated by the Muslims.
Bin Laden and al-Zawahiri have eluded capture since US-led forces toppled Afghanistan's Taliban government in 2001 after al-Qaida's 11 September attacks on the US.