The killing threatened to raise tensions amid a violent standoff between al-Sadr's Mahdi Army militia and the U.S.-backed Iraqi government.
In a statement Friday, al-Sadr blamed the United States and the Iraqi government for the death of Riyadh al-Nouri, the director of al-Sadr's office in Najaf. Al-Sadr urges his followers to be "patient."
Al-Nouri was gunned down as he drove home after attending Friday prayers in the adjacent city of Kufa, a police officer and a local Sadrist official said. Both spoke on condition of anonymity because they were not authorized to speak to the media.
Al-Sadr has his headquarters in Najaf, but the shrines in that city are dominated by a rival Shiite group and most of his followers are concentrated in Kufa.
Al-Nouri and a top al-Sadr lieutenant, Sheik Mustafa al-Yacoubi, were detained by American forces in April 2004 in the killing a year earlier of a moderate Shiite cleric, Sheik Abdul-Majid al-Khoei, in Najaf shortly after the U.S.-led invasion of Iraq. An arrest warrant was issued for al-Sadr himself but never served.
That along with the closing by U.S. authorities of al-Sadr's newspaper triggered a massive uprising that engulfed Shiite areas of central and southern Iraq. Several thousand people were killed before the rebellion was finally suppressed, and the two men were released in 2005.
Al-Sadr's spokesman in Najaf, Salah al-Obeidi, said the United States bore responsibility for Friday's killing because of its continued presence in Iraq. Al-Obeidi said the cleric appealed for calm and ordered his followers "not to be dragged into others' plots."
Police said al-Nouri was driving his car alone and had passed through two of their checkpoints before heading for the residential part of the city in which he lived. The gunmen were waiting for near his home, where no security forces were present.
An overnight curfew also was announced in the southern Shiite city of Hillah.
In other developments:
Meanwhile, sporadic clashes between Iraqi security forces and militia fighters broke out for a sixth day in the Mahdi Army strongholds of Baghdad's Sadr City and the southern port city of Basra.
And a rocket apparently aimed at the U.S.-protected Green Zone also fell short, crashing into a second-floor room and blowing a hole in the wall of the Palestine Hotel in central Baghdad. Police said three people were killed and seven wounded, mainly pedestrians on the street below.
U.S. airstrikes also killed 12 more suspected militants.
An unmanned drone fired on a group of gunmen carrying grenades and mortars overnight in Sadr City, killing six of them, the U.S. military said.
Armed drones are routinely used for long air patrols over the capital. They rely on their sensors to pick up militant activity during the night, and insurgents do not have air defenses capable of shooting down the slow-moving aircraft.
And the British military said a helicopter had hit a group of gunmen in the Hayaniyah district of central Basra overnight, killing six of them.
"They were positively identified as an active mortar team," British military spokesman Maj. Tom Holloway said.
The southern port city was the scene of fierce combat when Iraqi government forces launched a weeklong offensive against Shiite militias on March 25. British forces also took part in the fighting.
But that violence has ebbed. On Friday, authorities lifted a two-week ban on vehicle movement in Baghdad's mainly Shiite Shula neighborhood. A similar ban on vehicles in Sadr City district is scheduled to be lifted on Saturday.
Violence in Iraq had declined last year and early this year following a seven-month-old cease-fire by al-Sadr, an influx of American troops and a Sunni revolt against al Qaeda in Iraq.
But the recent government crackdown on the Mahdi Army has provoked fierce retaliation, underscoring the fragility of the security gains.
Separately, the U.S. military said Friday that the pullout of the five brigades that comprised last year's buildup of U.S. forces into Iraq is continuing with the redeployment of the 4th Brigade of the First Infantry Division back to Fort Riley in Kansas. The 4th Brigade was based in southern Baghdad, a district of about 1.2 million people.
All five surge brigades are due to return home by the end of July, leaving about 140,000 U.S. troops in Iraq.
Also Friday, a suicide car bomber struck a police checkpoint at an entrance to the Anbar province capital of Ramadi, killing three officers and wounding five others, police said.