The genes specifying chemotaxis, motility, and flagellar function in Escherichia coli are coordinately regulated and form a large and complex regulon. Despite the importance of these genes in controlling bacterial behavior, little is known of the molecular mechanisms that regulate their expression. We have identified a minor form of E. coli RNA polymerase that specifically transcribes several E. coli chemotaxis/flagellar genes in vitro and is likely to carry out transcription of these genes in vivo. The enzyme was purified to near homogeneity based on its ability to initiate transcription of the E. coli tar chemotaxis gene at start sites that are used in vivo. Specific tar transcription activity is associated with a polypeptide of apparent 28-kDa molecular mass that remains bound to the E. coli RNA polymerase throughout purification. This peptide behaves as a secondary sigma factor--designated sigma F--because it restores specific tar transcription activity when added to core RNA polymerase. The sigma F holoenzyme also transcribes the E. coli tsr and flaAI genes in vitro as well as several Bacillus subtilis genes that are transcribed specifically by the sigma 28 form of B. subtilis RNA polymerase. The latter holoenzyme is implicated in transcription of flagellar and chemotaxis genes in B. subtilis. Hence E. coli sigma F holoenzyme appears to be analogous to the B. subtilis sigma 28 RNA polymerase, both in its promoter specificity and in the nature of the regulon it controls.