Temperature is a key environmental cue for Yersinia enterocolitica as well as for the two other closely related pathogens, Yersinia pestis and Yersinia pseudotuberculosis. Between the range of 30°C and 37°C, Y. enterocolitica phase-varies between motility and plasmid-encoded virulence gene expression. To determine how temperature regulates Y. enterocolitica motility, we have been dissecting the flagellar regulatory hierarchy to determine at which level motility is blocked by elevated temperature (37°C). Here we report the cloning, DNA sequences, and regulation of the two main regulators of Class III flagellar genes, fliA (σF) and flgM (anti-σF), and a third gene, flgN, which we show is required for filament assembly. Identification of the Y. enterocolitica fliA and flgM genes was accomplished by functional complementation of both S. typhimurium and Y. enterocolitica mutations and by DNA sequence analysis. The Y. enterocolitica fliA gene, encoding the flagellar-specific σ-factor, σF, maps immediately downstream of the three flagellin structural genes. The flgM and flgN genes, encoding anti-σF and a gene product required for filament assembly, respectively, map downstream of the invasin (inv) gene but are transcribed in the opposite (convergent) direction. By using Northern blot analyses we show that transcription of both fliA and flgM is immediately arrested when cells are exposed to 37°C, coincident with the timing of virulence gene induction. Unlike S. typhimurium flgM− mutants, Y. enterocolitica flgM− mutants are fully virulent.