Singletary gave Balmer a pass Monday and Tuesday to deal with a "personal issue." Wednesday's absence was unexcused.
"I told Kentwan to be here this morning," Singletary said. "He's not here. He is under contract. The fact that he's not here, that speaks to me. What it says, I really don't know. As far as I'm concerned, I don't know what he's doing."
Balmer, San Francisco's first-round draft pick in 2008, is entering the third season of the five-year, $8 million deal that he signed as a rookie.
The defensive lineman from North Carolina has been a disappointment so far to the 49ers and was battling just to make the roster this summer. He has yet to start a game or record a sack in his two seasons with the team.
Balmer finished last season on injured reserve after missing San Francisco's final five games with a torn labrum. He declared himself healthy at the start of training camp this year, but he has fallen down the 49ers' depth chart behind starters Justin Smith and Isaac Sopoaga and veteran backups Ray McDonald and Demetric Evans.
Singletary said the 49ers would "probably have to bring in another player" along their defensive line. The team also is practicing without Aubrayo Franklin, who spearheaded San Francisco's 3-4 defensive scheme last season from his starting nose tackle position.
Franklin had a career season in 2009, and the 49ers designated him as their franchise player in March. Franklin missed all of San Francisco's offseason workouts and has remained absent during training camp as he seeks a new contract. He has yet to sign the team's one-year tender offer of $7.003 million and might not arrive until the end of this month.
Starting right end Justin Smith, the veteran leader of San Francisco's defensive line, said the unit already has adjusted to the absence of both Franklin and Balmer.
"We're moving along," Smith said. "Whatever the situation may be, in football, you're used to people going down, so it's no big deal. You just roll with what you've got."
Balmer's name recently has surfaced in news reports of an NCAA investigation at North Carolina, which he attended.
"I don't want to go there," Singletary said. "I'm done with Kentwan. I'm done. There is not going to be any phone calls (to Balmer). I don't want to say anything further than that. We're doing what we have to do. I don't want to talk anymore about something that's not here."
Copyright 2010 by The Associated Press