In a phone call to Libyan state television station, Gaddafi angrily denounced the rebels and said he would not surrender.
"We will not kneel!" he shouted in the phone call that appeared to also take state television by surprise. The sound was hastily adjusted to make it louder.
"We will not surrender: we only have one choice, to the end! Death, victory, it does not matter, we are not surrendering!" he shouted.
"We are stronger than your weapons, than your planes. The voices of the Libyan people are stronger than the sounds of explosions," he said, angrily calling the rebels who have risen up against him "bastards".
Minutes after he spoke, another explosion shook the capital as NATO apparently launched another strike.
Pro-Gaddafi loyalists also fired a round of celebratory gunfire after his speech, which lasted at least six minutes.
The date of the recording could not be confirmed, but his words suggested it was likely made today in the capital.
As he spoke, the sound of low-flying military craft could be heard whooshing through Tripoli again and Gaddafi quickly hung up.
Gaddafi has mostly been in hiding since NATO strikes in April targeted one of his homes. Libyan officials said one of his sons, Saif al-Arab, and three of his grandchildren were killed.