Tripoli, Libya (CNN) -- NATO's top official "strongly regretted" a fatal airstrike that may have mistakenly killed people it has pledged to protect, angering Libyan opposition leaders amid an increasingly frustrating campaign to oust Moammar Gadhafi.
NATO Secretary-General Anders Fogh Rasmussen called the incident unfortunate and said he regretted the loss of life.
"We are conducting operations in Libya in accordance with the U.N. Security Council resolution with the aim to protect civilians," he said. "This is also the reason why our aircraft target military equipment that could be used to attack civilians, but I can assure you that we do our utmost to avoid civilian casualties."
His comments came after the the deputy commander of the NATO operations refused to apologize
Gadhafi's forces attacked the western city of Ajdabiya on Friday with a barrage of artillery fire at the city's western gates. Ajdabiya has changed hands several times already, and rebels were forced to flee again in a war that is now viewed in some circles as unwinnable for the opposition, even with NATO air support.
NATO, meanwhile, was on the defensive Friday after reports of casualties apparently caused by the airstrike. British Royal Navy Rear Adm. Russell Harding said NATO forces may have hit rebel tanks near the eastern oil town of al-Brega on Thursday.
Opposition members said five people were killed when missiles struck a rebel formation on the eastern Libyan battlefront.
It was the second time NATO has been blamed for civilian deaths, Last week, opposition leaders said NATO airstrikes killed 13 civilians in the al-Brega area. NATO is investigating that strike as well.
"I'm not apologizing," Harding, the deputy commander of the NATO operation, said of the latest incident. "The situation on the ground is fluid, and we had no information the opposition forces were using tanks."
Harding said NATO had only recently learned that opposition forces had tanks. In the past, it was Gadhafi's tanks that had taken aim at civilians, he said.
"There's a lot of vehicles going back and forth," he said. "It is very difficult to distinguish who is operating the vehicles."
The airstrikes also injured 14 people, and an additional six are missing, said Gen. Abdul Fattah Yunis, a commander of the rebel forces.
Sorrow quickly turned to anger at a hospital where the wounded were taken, complicating matters for opposition fighters, already demoralized by the superior firepower of the Libyan army.
"NATO, NATO, NATO! They shouldn't hit the revolutionaries. We're helpless," one person screamed.
Ahmed Abu Bakr, a doctor who came to Libya from Germany to volunteer, said he never thought that he would be treating the wounded from friendly fire.
"I am very unhappy," he said. "They came here to help us, not injure us."
After the aerial attack Thursday morning, Gadhafi's troops pushed the rebels back, retaking territory and moving the front line farther east, Yunis said.
He said the rebels notified NATO of their tank movement and of their presence.
"There is no tension between us and NATO; this is a war situation, and we understand that mistakes are made," Yunis said.
Harding said Friday he did not feel NATO needed to strengthen communications with rebels.
"I have to be frank, it's not for us to improve communications. We have to see where civilians are being attacked and see if we can take action," Harding said.
A U.S. official with knowledge of the situation in Libya told CNN that CIA personnel remain in Libya to assess rebel strength and organization.
"They are the lead for anything that goes on," the official said.
The official said, however, that they still don't have a full understanding of the rebel structure and who is backing individual elements of their forces.
A few hours after the NATO strikes, civilians and rebels, fearing an approach by Gadhafi's forces, began retreating from Ajdabiya, with hundreds of civilian cars and trucks loaded with rocket launchers and ammunition headed out of town in the direction of the rebel stronghold of Benghazi, 100 miles away.
Ajdabiya was a ghost town Friday, raising questions about whether the outgunned rebel forces could prevail, and whether NATO has the correct strategy to help.
U.S. Gen. Carter Ham, who led the Libya mission before NATO took control, told lawmakers in Washington that the likelihood of rebel forces marching to Tripoli and ousting Gadhafi by military force -- even with NATO air power -- was "low."
Former CIA operative Robert Baer said NATO will have to put boots on the ground because "the no-fly zone is not working."
"Not a surprise to me that NATO bombed the rebel force," Baer told CNN. "We sort of got one foot in this, but not completely. The logic of this conflict is you have to put people on the ground."
The United States ratcheted up pressure Friday on Gadhafi financially by extending sanctions to five senior Libyan government officials and two entities controlled by Gadhafi's children.
The sanctions bar business transactions with those on the list and freeze their assets that fall under U.S. jurisdiction.
On the diplomatic front, a former U.S. lawmaker left Libya on Friday after failing to obtain a direct meeting with Gadhafi.
Curt Weldon said he is, however, carrying a letter from the Libyan leader to U.S. Secretary of State Hillary Clinton.
"I am disappointed that I did not get to sit down face to face with Colonel Gadhafi as promised, but I may have been able to get something even more significant -- a path to a resolution of this conflict," Weldon said in a statement on his departure. "Any time you are asked to play a part in advancing the cause of peace there is a moral obligation to say yes."
A former Republican U.S. House member from suburban Philadelphia, Weldon had previously been to Libya in his work as a congressman. He said his return this time with a small, private delegation was intended to convey the Obama administration's stance and reinforce the importance of an immediate cease-fire.
The African Union planned to meet Saturday in Mauritania to discuss engaging the opposing sides in the Libyan conflict, according to a statement from the South African government.
NATO has granted the committee permission to enter Libya and meet with Gadhafi in Tripoli, the statement said. They will also meet with opposition leaders in Benghazi on Sunday and Monday, it said.
Meanwhile, the World Food Programme said one of its humanitarian ships loaded with food, medical supplies and doctors has reached the besieged western city of Misrata, providing what it called a "lifeline" for trapped civilians.
CNN's Nic Robertson and Ben Wedeman contributed to this report.