Transcription of the late (Class 3) flagellar promoters in Salmonella typhimurium is dependent upon the flagellar specific sigma factor, sigma28, encoded by the fliA gene. sigma28-dependent transcription is inhibited by an anti-sigma factor, FlgM, through a direct interaction. FlgM can bind both to free sigma28 to prevent it from forming a complex with core RNA polymerase, and to sigma28 holoenzyme to destabilize the complex. A collection of fliA mutants defective for negative regulation by FlgM (fliA* mutants) were isolated. This collection included 27 substitution mutations that conferred insensitivity to FlgM in vivo. The distribution of mutations defined three potential FlgM binding domains in conserved sigma factor regions 2.1, 3.1 and 4 of sigma28. A subset of mutants from each region was assayed for FlgM binding and transcriptional activity in vitro. The results strongly support a multipartite interaction between sigma28 and FlgM. Region 4 mutations, but not region 2.1 or 3.1 mutations, interfered with the ability of FlgM to destabilize sigma28 from core RNA polymerase. We present refined models for FlgM inhibition of sigma28, and for FlgM destabilization of sigma28 holoenzyme.