Thomas E Blanton Jr and Bobby Frank Cherry are both former members of the Ku Klux Klan.
Mr Cherry's attorney, Mickey Johnson, said he was charged with eight counts of murder - two counts covering each of the four dead girls.
He said one count was for intentional murder and the other involved "universal malice" because the bomb was placed where it could have killed many more.
Both lawyers said their clients deny wrongdoing. Mr Cherry is said to be ill.
"He wants the world to know his story, and he thinks he'll be vindicated," said Mr Johnson.
Church killings
The two men are the only two living suspects in the bombing, which killed the girls at church on a Sunday morning.
It was one of the most shocking racial crimes of the civil rights era.
Church members were gathered for Sunday service on 15 September, 1963, when a dynamite bomb planted outside demolished a wall.
Eleven-year-old Denise McNair and three 14-year-olds, Cynthia Wesley, Carole Robertson and Addie Mae Collins were killed.
Moderate whites became more vocal in their opposition to segregation following the explosion, which came just months after police used dogs and fire hoses to confront black marchers led by the leading civil rights campaigner, Martin Luther King.
The initial investigation brought no charges, though the FBI named the four Ku Klux Klansmen as suspects.
An investigation in the 1970s resulted in the murder conviction of suspect Robert Edward Chambliss, who died in prison in 1985 while serving a life term.
A fourth suspect, Herman Cash, is dead.
After Chambliss' conviction, the case was reopened in 1980 and 1988, but no new charges were brought.
It was reopened yet again in 1997.
The bombing is also the subject of director Spike Lee's Oscar-nominated 1997 documentary, "4 Little Girls."