The flgM gene product has been shown to be a negative regulator of flagellin transcription in Salmonella typhimurium (K. L. Gillen and K. T. Hughes, J. Bacteriol. 173:2301-2310, 6453-6459, 1991; K. Ohnishi, K. Kutsukake, H. Suzuki, and T. Iino, Mol. Microbiol. 6:3149-3157, 1992). Mud-lac fusions to the flgM gene were isolated and used to characterize the regulation of flgM gene expression. Transcription of the flgM gene was decreased more than 30-fold in strains with the flagellar master regulatory genes, flhC and flhD, deleted. A class 2 flagellar defect caused a slight increase of flgM gene transcription unless a wild-type copy of the flgM gene was present, in which case transcription was decreased threefold. A deletion in the gene for the alternative sigma factor sigma 28 (FliA) caused a fourfold decrease in flgM expression. Insertional inactivation of a gene upstream of the flgM gene (flgA) in a fliA mutant strain caused transcription of the flgM gene to be decreased to a basal level. Northern (RNA) blot analysis confirmed the presence of two transcripts through the flgM gene, one which initiates upstream of the flgM gene and a second which initiates upstream of the flgA gene.