The broadcast also came after the Libyan government accused NATO bombing of killing more people in the oil town of Brega.
Gaddafi called the Western countries "cowards" in his minute-long message.
"You could kill my body but could not kill my soul which lives in the hearts of millions," Gaddafi said.
He also said a NATO airstrike Thursday that targeted his Bab al-Aziziya compound in Tripoli killed "three innocent journalist-civilians."
Italian Foreign Minister Franco Frattini said earlier Friday Gaddafi may have been hurt in NATO air raid and fled Tripoli, but this was later denied by a Libyan government spokesman.
Frattini said Tripoli's bishop had said Gaddafi was "probably wounded" but Italy had "no credible information" about Gaddafi's whereabouts and health conditions, according to the Italy-based ANSA news agency.
"International pressure has likely provoked the decision by Gaddafi to seek refuge in a safe place," the foreign minister told reporters.
Shortly after the report, Libyan government spokesperson Musa Ibrahim denied Gaddafi was hurt, according to Al-Arabiya TV.
He said these reports were made only to weaken the morale of the Libyan people.
Rumors that Gaddafi was dead have been circulating since his youngest son, Saif al-Arab Gaddafi, and three of his grandchildren were killed in a NATO bombing almost two weeks ago.
Gaddafi made his first TV appearance since that attack on Wednesday. The video footage showed him meeting with several tribal leaders, which was seen as the Libyan government's attempt to dispel the rumors.
Libya's state TV also reported Friday at least 16 people were killed in a NATO air strike at Brega, a key oil town in the eastern part of the country, which is still reportedly controlled by pro-Gaddafi forces. The television said the air raid targeted a guest house in the city.
The NATO-led alliance, responding to the civilian deaths, said in a statement released in Brussels on Saturday it attacked a military command and control center, 750 km southeast of the capital.
Also on Friday, Libyan rebels held a meeting attended by delegates from 15 cities around the country, a move seen as an attempt to scuttle regional support for Gaddafi.
The 15 cities includes both those captured by the opposition and those still in the hands of pro-Gaddafi forces, including Sirte, the hometown of Gaddafi.
Besides courting domestic support, the rebels' National Transitional Council (NTC) is also seeking additional international recognition.
NTC chief diplomat Mahmoud Gibril, who is now in the United States, met with U.S. National Security Adviser Tom Donilon at the White House Friday.
Donilon said Washington viewed the NTC as a legitimate and credible interlocutor of the Libyan people and reiterated U.S. President Barack Obama's call for Gaddafi to leave immediately, the White House said in a statement.
The White House also said Donilon and Gibril discussed how the United States and the coalition could provide additional support to the NTC, according to the statement.
However, White House spokesman Jay Carney said Friday he did not anticipate formal U.S. recognition of the NTC as the legitimate representative of the Libyan people after the meeting.
"The question of recognition is one of many policy issues still under review, and we're continuing to assess the capabilities of the NTC as we deepen our engagement with the opposition," he told reporters at the White House.
He said the White House was discussing with Congress access to blocked Libyan government assets in the United States, valued at some 30 billion U.S. dollars, for humanitarian purposes in Libya.
Russia also supports release of the assets for humanitarian aid, Russian Foreign Ministry spokesman Alexei Sazonov told a news briefing Friday.
Sazonov said such humanitarian aids should not be politically motivated and should cover all of the nation's population.
He said these actions should be approved by the UN Security Council and international organizations such as UN High Commissioner for Refugees should monitor the use of these aids.
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