We have studied the molecular mechanism of the negative regulation by flgM of the late operons of the flagellar regulon of Salmonella typhimurium. A 7.8 kDa protein that was identified as the flgM gene product was purified to homogeneity; its amino-terminal sequence was identical to the deduced sequence except for the lack of the initiating methionine. The purified FlgM repressed transcription from the fliC promoter, one that is activated by the sigma factor, FliA (σF). No DNA-binding activity was detected in FlgM. Chemical cross-linking experiments showed that the purified FlgM bound to σF and disturbed Its ability to form a complex with RNA polymerase core enzyme. These results indicate that FlgM is a novel type of negative regulator that probably inactivates the flagellum-specific sigma factor through direct interaction, i.e. it is an anti-sigma factor.