Al-Zawahiri, and the American convert identified as Adam Gadahn, called for Americans to convert to Islam in the 41-minute video posted on an Islamist website just nine days before the fifth September 11 anniversary.
The FBI says that the man who appeared next to al-Zawahiri attended al-Qaeda training schools in Pakistan and served as a translator for al-Qaeda.
It was the second time Gadahn has appeared with al-Zawahiri. The first was on a video posted on July 7, marking the one-year anniversary of London underground bombings, when Gadahn said no Muslim should "shed tears" for Westerners killed by al-Qaeda attacks.
Gadahn and al-Zawahiri did not appear together in the footage. Gadahn spoke with his face uncovered, resembling the FBI photos, with his name subtitled in English and Arabic as "Azzam the American".
"We invite all Americans and unbelievers to Islam," Gadahn said, dressed in a white robe and turban with a long, thick black beard and a computer terminal in the background.
Wanted by FBI
Gadahn, a 28-year-old from California who converted to Islam, is wanted by the FBI in connection with possible terrorist threats against the US, though the agency says it has no information linking him to any specific terrorist activities.
Gadahn spoke for much of the video, saying he wanted to correct the incorrect image Americans have of Islam.
Gadahn (bottom, 2nd from L) is
wanted by the FBI for threats
He described the "West" as the civilisation which enslaved Africa, slaughtered native Americans and bombed Tokyo, Falluja and used nuclear bombs on Hiroshima and Nagasaki.
He said it shows more concern for archaeological sites, like statues of Buddha destroyed by Afghanistan's former Taliban rulers, "than it shows of the people of Afghanistan and Iraq".
He said: "Ignorance of Islam causes the people of the West to rapturously applaud when Israel perpetrates wholesale slaughter of Muslims in Lebanon and Palestine and leads them to give their consent to the atrocities that governments commit in Afghanistan, Iraq and elsewhere in the Muslim world."
The video, issued by al-Qaeda's production wing As-Sahab, had been advertised on militant websites for several days before it appeared on Saturday.
Besides the July 7 video, Gadahn is believed to be a masked figure who appeared in two unofficial videos given to the American television network ABC in Pakistan in 2004 and a few days before September 11, 2005.
In the 2005 tape, the speaker threatened new terror attacks in Los Angeles and Melbourne, Australia.
The 2004 tape praised the September 11 attacks and said a new wave of attacks could come at any moment.