National Nuclear Security Administration spokeswoman Julie Smith said the latest shipment of three kilograms of highly enriched uranium (HEU) had been extracted over a two-day period this week from Gadhafi's Tajura research reactor.
The facility is near the Mediterranean coast about 16 kilometres east of Tripoli, Libya's capital.
"We had to wait to announce it to make sure it had arrived safely", Ms Smith said. The other shipment of 17 kilograms was returned to Russia in 2004.
The agency's announcement said the operation "is part of a multistep project to remove all Russian-origin HEU material from Libya".
The agency said in a news release that the fresh highly enriched uranium, which could have been used in nuclear weapons, would be blended down to safer low enriched uranium and returned.
Libyan leader Gadhafi renounced weapons of mass destruction and all links to terrorism in late 2003.
A year later, in September 2004, the United States began easing sanctions against Libya, renewing commercial air links and restoring Libyan access to A$1.7 billion that had been frozen.
In making that announcement, Scott McClellan, then the White House spokesman, said Libya had facilitated removal of "all significant elements of its declared nuclear weapons program", destroyed its chemical munitions and removed highly enriched uranium and equipment for uranium enrichment.
Russia announced a week earlier that Russia had undertaken to recover highly enriched uranium it had shipped to civilian research reactors to reduce chances that the material might be obtained by terrorists. Among countries mentioned was Libya.
Approximately 189 kilograms of highly enriched uranium had been returned to Russia in 13 shipments from Serbia, Romania, Bulgaria, Uzbekistan, Latvia, the Czech Republic and Libya.
It said experts from the International Atomic Energy Agency, Libya, Russia and the United States handled the recovery operation under the nuclear security administration's Global Threat Reduction Initiative.