The Khazali brothers were formerly ranking members of al-Sadr's Shia movement.
Qais Khazali served as its spokesman in August 2004 during battles between US troops and al-Sadr's Mahdi Army militia.
Al-Sadr officials said Thursday that Qais had split from the Mahdi Army and was operating on his own, amid rumours he had received Iranian backing.
On January 20, men disguised in US army uniforms attacked a provincial security building in the Karbala province of central Iraq during a visit by American troops to their Iraqi counterparts.
One US soldier died during the assault and four were captured. Their bodies were later found in the attackers' abandoned vehicles.
The raid was one of the most sophisticated and carefully planned attacks on US troops in the four-year war.
Some reports speculated complicity between Iraqi officials and the attackers, while some internet reports alleged Iranian involvement.
The Pentagon regards al-Sadr's Mahdi Army as the most dangerous faction in Iraq's sectarian violence.
In recent months more than 700 members of his movement have been arrested.
Iraq's Shia-led government insists that al-Sadr supports a new security plan designed to end violence between Sunni and Shia factions, despite a US claim that he has fled to Iran.
The announcement of the Khazali arrests comes a day after Ahmed Shibani, a senior aide to al-Sadr, was released from the custody of US forces on the instruction of Nuri al-Maliki, Iraq's prime minister.
Shibani had been held for two-and-a-half years after being arrested by US forces on suspicion of carrying heavy weapons.