Ayman al-Zawahiri, al-Qaeda's number two, has warned that Osama Bin Laden will continue to "terrify" the US from beyond the grave.
The statement was posted on Jihadist websites.
It comes five weeks after US forces killed Bin Laden, and less than a week after another prominent figure, Ilyas Kashmiri, died in a US drone attack.
This is the first time al-Zawahiri has spoken publicly since Bin Laden's death.
It remains to be seen who - if anyone - will become the new, pre-eminent leader of the world's most notorious militant group.
Al-Zawahiri notably omits to refer to a new leader for al-Qaeda, despite speaking for 28 minutes in the new video.
Most of it is taken up with a eulogy to Bin Laden.
"The sheikh has departed, may God have mercy on him, to his God as a martyr and we must continue on his path of jihad to expel the invaders from the land of Muslims and to purify it from injustice," Zawahri says.
"Today, and thanks be to God, America is not facing an individual or a group, but a rebelling nation, which has awoken from its sleep in a jihadist renaissance."
Speaking in measured tones, al-Zawahiri denounces the Americans for burying his former leader at sea.
He also backs the wave of Arab unrest and calls for Sharia Islamic law to be applied in Egypt.
And he warns Libyans that Nato's bombing campaign seeks to replace Muammar Gaddafi with a Western-backed tyrannical regime.