British Tornado jets fired precision-guided missiles at a large bunker in Libyan leader Col Muammar Gaddafi's hometown of Sirte, officials have said.
The aircraft took off from RAF Marham in Norfolk on a long-range strike mission on Thursday night.
The Libyan rebels are building up their forces east of Sirte, in preparation for a major assault. They continue to face unexpectedly stiff resistance.
Rebel leaders are meanwhile calling on other states to unfreeze Libyan assets.
The UN has already agreed to release $1.5bn (£1bn) in funds, which had been frozen under Security Council sanctions at the beginning of the uprising in February, to help with immediate humanitarian needs.
But South African President Jacob Zuma said the African Union (AU) would not yet recognise the rebel National Transitional Council (NTC) as the sole representative of the Libyan people until hostilities ended.
"If there is fighting, there is fighting. So we can't stand here and say this is the legitimate [government] now. The process is fluid. That's part of what we inform countries - whether there is an authority to recognise," he said.
Later, the AU called for an inclusive transitional government to be formed.
In a statement issued on Friday, the UK Ministry of Defence said "a formation of Tornado GR4s... fired a salvo of Storm Shadow precision-guided missiles against a large headquarters bunker" in Sirte.
The bunker housed a command and control centre. There is no indication that Col Gaddafi was in Sirte, which is 250 miles (400km) east of Tripoli, or in the bunker itself at the time of the attack.
"It's not a question of finding Gaddafi, it's ensuring the regime does not have the capability to continue waging war against its own people," UK Defence Secretary Liam Fox told the BBC.
"The attack that we launched on the bunker in Sirte last night was to make sure that there was no alternative command and control should the regime try to leave Tripoli."
Nato warplanes also targeted 29 vehicles mounted with weapons near Sirte and bombed surface-to-air missile facilities near Tripoli, the alliance said at a daily briefing in Brussels.
Meanwhile, the rebels are building up their forces around the oil port town of Ras Lanuf, preparing for an assault on Sirte, about 250km (155 miles) along the coast to the west.
They had to withdraw from positions nearer Sirte to put themselves out of the range of Grad rockets being fired by Gaddafi loyalists.
The BBC's Paul Wood, who is with the rebels, says their mood is still buoyant, despite running into unexpectedly stiff resistance.
Rebel commanders think the fighting on the road to Sirte could last another three or four days, our correspondent says.
The National Transitional Council has begun moving to Tripoli, although many senior figures remain in the eastern stronghold of Benghazi.
Spokesman Ali Tarhouni said the NTC's chairman, Mustafa Abdul Jalil, would arrive as soon as the security situation permitted.
He called on forces still loyal to Col Gaddafi to surrender.
"Put your weapons down and go home. We will not take revenge. Between us and you is the law. I promise you will be safe," he said.
Speaking in Istanbul, the head of the NTC's cabinet, Mahmoud Jibril, said the uprising could fall apart if funds were not forthcoming quickly.
"The biggest destabilising element would be the failure... to deliver the necessary services and pay the salaries of the people who have not been paid for months," he said. "Our priorities cannot be carried out by the government without having the necessary money immediately."
In Tripoli, there was continued fighting near the international airport. Several aircraft were reportedly destroyed by rocket fire.
A rebel commander told the Associated Press the airport was largely under rebel control, but loyalists were shelling it from a nearby military base once controlled by Col Gaddafi's son, Khamis.
There was also fighting on Friday in the capital's southern Abu Salim district, one of the last areas loyal to Col Gaddafi.
A Scottish nurse working at the hospital in Abu Salim, Karen Graham, told the BBC they were "overwhelmed" with casualties.
"All the staff were just doing the best we can, but we were literally inundated," she said. "We'd just clear one lot of casualties and the next lot would be getting brought in. Our theatre just couldn't cope... This is the first time we've had such a vast number of people in."
Dozens of decomposing bodies were piled up at one hospital in the area, AP reported. One room had 21 lying on stretchers, while 20 more were left in a courtyard. It was not clear who killed the men, or when they died.
Human rights group Amnesty International says it has evidence that both pro-Gaddafi forces and rebels abused detainees in their care.
Guards loyal to Col Gaddafi raped child detainees at Abu Salim prison, Amnesty said. It also accused rebels of beating prisoners, including a boy conscripted by Gaddafi forces who surrendered to the rebels at Bir Tirfas.
The UN is to investigate reports of summary killings and torture through its existing commission of inquiry on Libya.
"We urge all those in positions of authority in Libya, including field commanders, to take active steps to ensure that no crimes, or acts of revenge, are committed," UN spokesman Rupert Colville told Reuters.
The UN has previously said some military action in Libya could amount to war crimes or crimes against humanity.
The BBC's Rupert Wingfield-Hayes visited a hospital in the Mitiga district of Tripoli which had received the bodies of 17 rebel fighters.
Doctors said the group had been prisoners of Gaddafi troops in Tripoli and were tortured and killed as the rebels seized the capital earlier this week.
Dr Hoez Zaitan, a British medic working at the hospital, said about half the bodies had bullet wounds to the back of the head while others had disfiguring injuries to their limbs and hands.
He said the bodies had been examined for possible evidence to be used at a war crimes tribunal.