Anders Fogh Rasmussen, like the NATO military commander, Rear Adm. Russell Harding, did not apologize for the rebel deaths.
"I strongly regret the loss of life," Rasmussen said. "The situation on the ground is very fluid. We have seen in the past that tanks have been used by the Gadhafi regime to attack civilians."
During a news conference at NATO's operational headquarters in Naples, Italy, Rear Adm. Russell Harding said the alliance wasn't told rebels were using tanks when the attack occurred Thursday, The New York Times reported.
Reports differ on the death toll, ranging from four to 13.
Gen. Abdul Fattah Younes, commander of the rebel army fighting to oust leader Moammar Gadhafi, said a column of tanks deployed by the insurgents for the first time was the target of a "fierce attack."
"It is likely it is NATO by mistake," Younes said. He said rebels notified NATO in advance the tanks were headed to the front.
Harding called movement in the area "extremely fluid" at the time of the attack.
"I am not apologizing. The situation on the ground was and remains extremely fluid and until yesterday we did not have information" that the rebels planned to deploy tanks, Harding said.
"Our role is to protect civilians and tanks have been used to threaten civilians" in other parts of Libya, particularly in the western port of Misurata where forces loyal to Gadhafi have used tanks against rebels, he said.
Thursday's incident marked the second time in less than a week that NATO warplanes struck a rebel target. Airstrikes killed 13 rebels in the same region Saturday.
Harding denied responsibility for a strike on the Sarir oilfield, accusing Gadhafi forces of being behind the attack, The Daily Telegraph reported.
"There was no NATO strike in that area," he said.
In Tripoli, government spokesman Musa Ibrahim said NATO airstrikes hit at least three military academies, the Times reported.