In an interview with Swedish newspaper Expressen, Mustafa Abdel-Jalil, a confidant of Gadhafi's who resigned his post amid this week's anti-government protests and civil strife, said, "I have proof that Gadhafi gave the order about Lockerbie."
Some family members of the victims said the admission vindicated what they had long believed and called on the U.S. government to officially acknowledge that the 1988 bombing over Lockerbie, Scotland, was state-sponsored terrorism.
"There can no longer be any doubt about the involvement Gadhafi had in murdering American citizens," said Bert Ammerman, whose brother, Tom, died in the attacks. "He ordered the bombing of Flight 103. I challenge President Obama to respond accordingly. There must be justice."
Abdel-Jalil told the paper that Gadhafi ordered Abdelbaset al-Megrahi, a suspected intelligence agent and the only man convicted of the bombing that killed 259 people aboard the plan and another 11 on the ground, to carry out the attack.
"To hide it, [Gadhafi] did everything in his power to get al-Megrahi back from Scotland," Abdel-Jalil told the paper.
Megrahi was released from a Scottish prison in 2009 on compassionate grounds because he had cancer and claimed he had only weeks to live. His release caused an uproar in the West, with some family members of victims accusing the British government of cutting a deal with Libya to secure lucrative oil contracts.
Megrahi returned to Tripoli to a hero's welcome. He is still alive and living in a seaside mansion provided by the government.
U.S. officials have long suspected or known Gadhafi was likely behind the attack, but were unable to accuse a head of state of murder without significant proof and the political will to call for his arrest, said Brad Garrett, a former FBI agent and ABC News consultant.
"At the time of the investigation, FBI agents found substantial evidence that they believed Gadhafi was linked directly to the bombings but never proved it," Garrett said.
"If you point a finger at a head of state, you've really got to have it down pat. The level of evidence available was just not there," he said. "Just about everyone on the joint FBI and Scottish task force believed Gadhafi was behind the attacks."
Since the late 1990s, Gadhafi has been trying to revamp his image on the international stage, making small concessions to successive U.S. presidents and cooperating openly with Europe.
Without acknowledging any wrongdoing, his government has paid settlements to family members of the victims. In 2001, he dismantled his nuclear arms program, normalizing relations with the United States.
The latest accusations by Abdel-Jalil have again raised the ire of some victims' family members, most of whom are Americans.
"Gadhafi has been accused of an act of war on the U.S.," Bert Ammerman said.
"Bush 41 [George H. W. Bush] said in 1989 if there was any evidence of state-sponsored terrorism, he would act militarily. The Obama administration must react to this statement now," he said.
State Department spokesman P.J. Crowley said he is "in no position to corroborate" the story Abdel-Jalil told Expressen.
Asked by ABCNews.com whether the information would lead to the United States' officially accusing Gadhafi of state-sponsored terrorism, Crowley said: "You know, that is an interesting story. Beyond that, I can't comment at this point."
ABC News' Kirit Radit contributed to this report.