An official at the hospital in Kufa said on Friday three people had died in gunfire and mortar shelling in the town, while eight were wounded.
In Najaf, five kilometres southwest of Kufa, two were killed and six hurt, hospital staff said.
Two US soldiers were also wounded in the clashes after their Humvee military vehicle came under fire from militia fighters in Kufa, a US military spokeswoman said.
US forces and militiamen loyal to Shia leader Muqtada al-Sadr had struck a tentative truce on Thursday, but fighting broke out again early on Friday.
Al-Sadr, who has led an uprising against US forces throughout southern Iraq for the past two months, said he would disarm his militia and withdraw from Najaf as long as US forces also withdrew.
But members of al-Sadr's al-Mahdi army fired rocket-propelled grenades when three US tanks advanced towards the centre of Kufa, reported an Iraqi journalist at the scene.
A Reuters correspondent heard sporadic gunfire around al-Sadr's home mosque at Kufa, just outside Najaf and US tanks cordoned off the area.
The Shia leader usually preaches the Friday noon sermon at the Kufa mosque.
However, al-Sadr did not turn up for the prayers. An aide confirmed the Shia leader would not appear at the Kufa mosque and said he did not know his whereabouts.
Several thousand worshippers had packed the mosque and
grounds outside it to show support for al-Sadr.
Thousands of men, including heavily armed militia, were seen in the back streets of Kufa and Najaf after occupation soldiers blocked off the roads between the two cities.
Al-Sadr offered to withdraw fighters from Najaf if US occupation forces retreat from the city where hundreds of people have been killed.
The agreement, struck via local clerics with no direct involvement of the US military, also involved US authorities holding off arresting al-Sadr in connection with the murder of a rival Shia leader in April last year.
A Shia holy shrine, the Imam Ali mosque was also damaged in the fighting this week.