Jets screeched overhead as massive explosions and tank and machine-gun fire boomed through the city and smoke engulfed its historic centre, home to the shrine of Hazrat Ali (RA).
In Baghdad's district of Kadhimiyah and the British-patrolled southern oil city of Basra, thousands of people protested against US attacks on holy cities, held aloft pictures of Moqtada Sadr and denounced Prime Minister Iyad Allawi.
Thousands of US forces, backed by Iraqi police and national guard, mounted a pincer movement to trap Moqtada Sadr's fighters in the heart of the city, before going on to raid the militia leader's empty home.
Iraqi and US troops sealed approaches to the mausoleum, as hundreds of terrified residents, urged on by attacking forces and the city's mosques, fled through the dusty streets.
"Leave the city. Help coalition forces and do not fire at them," one announcement instructed in Arabic. "We are here to liberate the city."
Armed militiamen fanned out into the deserted plaza outside the shrine as mosques urged the Mehdi Army to defy the onslaught and defend the city.
By dusk, one militiamen had been killed and 25 wounded, while one civilian was killed and three others injured, said the clinic inside the shrine.
The government said the joint offensive would continue, demanded the Mehdi Army evacuate the holy shrine but pledged US forces would not be allowed to enter the sacred mausoleum.
"The operations are continuing... and will continue until the militia is forced out or they surrender," Defence Minister Hazem al-Shalan told a press conference in Baghdad.
In Najaf, the militia, still in control of the area around the shrine, vowed to fight until the bitter end.
"We are ready to fight until the last drop of blood if this is what the Americans want," said Sheikh Ali al-Sumeisim.
Various efforts were underway to defuse the stand-off. The government's pointman on security, Muwafaq al-Rubaie, headed to Najaf in a bid to meet with Sadr and end the assault.
The Najaf office of Iraq's revered Shia spiritual leader Grand Ayatollah Ali Sistani also said it was working with all sides for peace.
Envoy Hussein al-Shahrastani said that if Sistani had predicted the scale of the crisis he would never have left for medical treatment in Britain.
"We ask all sides to immediately renounce arms to save Muslim blood and the sanctity of the city," he said.
But Iraq's top Sunni body warned the security forces against supporting the US military.
Early on Thursday, Najaf deputy governor Jawdat Kadam Najem al-Kuraishi and half of the 30-member provincial council resigned in protest against US "terrorist operations" and the "hasty US invasion" of Najaf.
Twenty-four hours of nation-wide fighting, mostly in the Shia south and Sadr's Baghdad stronghold, has claimed 165 lives and wounded 594, the health ministry said.
A British soldier was later killed in Basra when his patrol was struck by a home-made bomb - the second British soldier to die in 72 hours in Iraq.
84 KILLED IN KUT: Further north, in Kut, which fell briefly to the Mehdi Army in Sadr's spring uprising against the US-led occupation, heavy overnight US bombing killed 84 people and wounded 176, medics said.
US planes pounded the southern Al-Shakia district, a densely populated Mehdi Army stronghold, but medics said many of the dead were women and children.
The bombs also flattened the local office of Sadr's movement, which a partisan said was empty at the time.
"We never expected to see so many bodies. Our hospital beds are full and many wounded are still lying in the corridor," said doctor Khader Fadal Arar.
The Mehdi Army then attacked a police station, killing one officer and wounding nine, while Shalan said 400 militiamen had been killed, captured or wounded in Kut.
In the Baghdad militia stronghold of Sadr City, two people were killed after they attacked a US patrol, the military said.
The uprising, which has fanned out across Shia cities south of Najaf and forced the closure of a southern oil pipeline, has halved Iraq's crude exports and led to losses of about 60 million dollars, the government said.
Fighting in Najaf and heightened Mehdi threats against oil infrastructure saw world oil prices soar to all-time highs.
A shadowy Shia militant group also threatened to kill all those working with British troops in the region. It was not clear if the group had any direct links with the Mehdi Army.
Meanwhile, two US marines were killed when a helicopter crashed in the volatile north-western province of Al-Anbar late on Wednesday, just hours after a US air strike on the Sunni Muslim insurgent bastion of Fallujah.
In Baghdad, US planes roared overhead as a sign of force when Mehdi Army militia attacked a police station in the centre of the capital and police called for US back-up, the militia said.