In an audio message aired by state television, the longtime ruler said he will continue to resist in the face of Western alliance attacks.
Gaddafi said the attacks will not frighten his people, adding that throughout history Tripoli has resisted such wars.
This is Gaddafi’s first message since May 19 when state television showed footage of him holding talks with a senior official.
In another audio message broadcasted on state television on May 13, Gaddafi said that he was in a place where NATO bombs could not reach.
The latest developments come as NATO continues to conduct sorties over Libya since the military alliance assumed control of a campaign to impose a no-fly zone over Libya, and save civilian lives in late March.
The US and NATO have unleashed a punishing, UN-mandated offensive against embattled Gaddafi to pressure him into relinquishing power.
However, the NATO airstrikes have killed scores of civilians as well as revolutionaries.
NATO has recently stepped up its air raids over Libya with the introduction of French and British attack helicopters.
NATO fighter jets have been pummeling the Libyan regime’s military installations, communication centers and armories.
Fighting also continues on the ground between pro-Gaddafi forces and Libyan revolutionary forces.
Libya has been the scene of fierce fighting between pro-Gaddafi troops and anti-regime forces since mid-February.
Revolutionary forces want an end to Gaddafi’s decades-long rule