Usama bin Ladin's right hand man, Ayman al-Zawahri is regarded as the Islamist network's second in command.
Al-Qaida's number-two urged all Muslims in the Asian country to reject Islamabad's cooperation with US forces and throw out the unelected military dictatorship of President Pervez Musharraf.
He also urged the Pakistani military to "disobey orders. I call on Muslims in Pakistan to get rid of their government which is working for Americans."
The seven minute broadcast on Thursday made no reference to the Madrid train bombings on 11 March but did mention the Pakistani offensive against tribal Taliban sympathizers in South Waziristan that began earlier in the month.
A key US ally in the "war on terror", Islamabad has been waging a bloody campaign for the last fortnight.
Paramilitaries have attempted to root out Taliban and al-Qaida sympathisers on its Afghan border at the same time as US forces are sweeping along on the other side of the frontier.
The US want al-Zawahri and bin
Ladin dead or alive
Pakistani officials said last week the fierce defence tribesmen were putting up suggested they were protecting a "high-value target" - perhaps al-Zawahri.
But the military later said that was unfounded "guesswork".
Islamabad has faced strong opposition demands to stop the offensive in the lawless South Waziristan tribal region waged with some 5000 troops.
But the military dictatorship says it will go on until all tribal resistance is eliminated. Scores of people have been killed.