Khamis, the feared military commander and son of Libyan leader Moamer Kadhafi, was shown on state television on Wednesday, days after rebels reported his death.
Tripoli denied the claim at the time, but a rebel spokesman on Wednesday insisted that Khamis was dead.
The broadcaster showed footage of what it said was a visit on Tuesday by the uniformed 28-year-old, Kadhafi's youngest son, to a hospital to meet "victims of NATO raids."
The date of the recording could not be confirmed. If genuine, it would be the first time Khamis had been seen in public since Friday, when rebels said a NATO strike on the western town of Zliten killed 32 people, including Khamis.
Kadhafi spokesman Mussa Ibrahim said at the time the claim was untrue, claiming it was "dirty lies to cover the murder of civilians" in Zliten.
Khamis trained at a Russian military academy and commands the eponymous and much-feared Khamis Brigade, one of the regime's toughest fighting units.
Reacting to the television report, rebel spokesman Abudulah Kabir said in the city of Misrata that the insurgents were sure Khamis was dead.
"The military council has confirmed he has died; we are sure," he said.
Meanwhile, US diplomats are visiting several African countries as part of efforts to urge leaders to press Kadhafi to leave power immediately, officials in Washington said on Tuesday.
Several African states, having benefited financially from Kadhafi's policies, have been reluctant to call for him to step down, and have criticised the NATO-led military campaign in Libya.
Gene Cretz, the US ambassador who left Tripoli before Kadhafi launched his bloody crackdown on the opposition in February, and Principal Deputy Assistant Secretary of State Donald Yamamoto arrived Monday in Addis Ababa, headquarters of the African Union, State Department deputy spokesman Mark Toner said.
They "are in Africa to meet with African Union members to discuss the crisis in Libya and the need for Kadhafi to relinquish power now," he told AFP.
They also met Mahmud Jibril, leader of Libya's opposition National Transitional Council (NTC), who was visiting Ethiopia.
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In Brussels, the European Union slapped new sanctions on the Kadhafi regime, targeting two "economic entities" linked to human rights abuse, EU foreign policy chief Catherine Ashton said.
An asset freeze and visa ban were extended to "two further entities closely linked to the perpetrators of the serious human rights abuses in Libya, given the gravity of the situation," Ashton said in a statement.
She gave no further details but a French official said on Tuesday the restrictions would target Al-Sharara, which operates in the oil sector, and an administrative organisation linked to Kadhafi.
Six port authorities, 49 entities and 39 people are now subject to a freeze of their funds and financial resources in the EU, Ashton said.
In addition, the same 39 persons, which include Kadhafi and several family members, are banned from entering the EU.
With Britain being one of the key powers engaged in efforts to get Kadhafi to step down, the Libya regime lashed out at Prime Minister David Cameron, calling him to resign over the riots that have hit England over the past few days.
"Cameron and his government must leave after the popular uprising against them and the violent repression of peaceful demonstrations by police," Libya's JANA quoted Deputy Foreign Minister Khaled Kaaim as saying.
"Cameron and his government have lost all legitimacy," he said.
"These demonstrations show that the British people reject this government which is trying to impose itself through force."
Kaaim called on the "UN Security Council and the international community not to stay with its arms crossed in the face of the flagrant violation of the rights of the British people."
Nightly riots which began in London and quickly spread to other major cities have gripped Britain since Saturday when an angry crowd marched to demand justice after a 29-year-old man was shot dead by police.
On Tuesday, the Libyan regime also said world powers would be held responsible for the "ugly massacre committed by NATO" on the village of Majer, where 85 people were killed, Libya's official JANA news agency reported.
Thirty-three children, 32 women and 20 men from 12 families were killed in the "massacre," spokesman Mussa said.
NATO insisted the raids were "legitimate" and said it had no evidence of civilian deaths.