"We have no information to indicate that he is anywhere else than in Pakistan," Pentagon spokesman George Little was quoted as saying about the 60-year-old Egyptian physician-turned-militant leader who succeeded Osama bin Laden.
Little's comments came after Zawahiri apparently posted a new message coinciding with the 10th anniversary of the 9/11 terror attacks.
The hour-long video, posted on Monday on jihadist websites, includes an audio broadcast purportedly from al- Zawahiri and a video message from bin Laden, recorded before his death in May in Pakistan.
Al-Zawahiri''s speech lauds the Arab Spring as having "liberated thousands of the members of the Islamic movement, who were imprisoned by direct orders from America."
"The popular revolutions are a form of defeat for the US, just as the 9/11 attacks and its alleged lack of success in Afghanistan and Iraq were also defeats," Zawahiri, who was elevated as the chief of al-Qaeda in June this year, said.
The uprising in a number of Arab nations, including Egypt, Tunisia and Libya, has also "liberated the Arab people from handcuffs of fear and terror," the message says.
The bin Laden video is apparently the same footage that was released by the US government after the al-Qaeda was killed in a raid by American commandos in Pakistan, but this one includes his audio as well.
The version issued by the Pentagon, after it was found in bin Laden's Abbottabad compound, was silent.