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Ayman al-Zawahri spoke in a 22-minute audio message posted on the Internet.
"The strike by our brothers in Guantanamo reveals the real odious and ugly face of America," he said. Some of the 166 prisoners there began a hunger strike earlier this year to protest conditions and their indefinite confinement.
"We pledge God that we will spare no efforts to set them free along with all our prisoners, on top of them Omar Abdel Rahman, Aafia Siddiqui, Khalid Sheikh Mohammed and every oppressed Muslim everywhere," he said.
Militants have in the past tried to free Abdel Rahman, a blind Egyptian sheik convicted of plotting to blow up New York City landmarks, and Siddiqui, a Pakistani scientist convicted of shooting at two U.S. soldiers in Afghanistan, by offering to free hostages in exchange.
Khalid Sheikh Mohammed, the admitted mastermind of the 9/11 terrorist attacks, is at Guantanamo. The other two are at civilian prisons in the United States.
The message's authenticity could not be independently confirmed but the message was posted on a militant website commonly used by al-Qaida.
Al-Zawahri's last message, urging Sunni Muslims to devote their lives, money and expertise to battling Syria's President Bashar Assad, was delivered in June. Assad's regime is dominated by members of a Shiite offshoot sect.