The alternative sigma factor, sigma D, activates the expression of genes required for chemotaxis and motility in Bacillus subtilis, including those encoding flagellin, hook-associated proteins, and the motor proteins. The sigma D protein is encoded in a large operon which also encodes the structural proteins for the basal body and homologs of the enteric CheW, CheY, CheA, and CheB chemotaxis proteins. We report the identification and molecular characterization of a novel chemotaxis gene, cheV. The predicted CheV gene product contains an amino-terminal CheW homologous domain linked to a response regulator domain of the CheY family, suggesting that either or both of these functions are duplicated. Transcription of cheV initiates from a sigma D-dependent promoter element both in vivo and in vitro, and expression of a cheV-lacZ fusion is completely dependent on sigD. Expression is repressed by nonpolar mutations in structural genes for the basal body, fliM or fliP, indicating that cheV belongs to class III in the B. subtilis flagellar hierarchy. The cheV locus is monocistronic and is located at 123 degrees on the B. subtilis genetic map near the previously defined cheX locus. A cheV mutant strain is motile but impaired in chemotaxis on swarm plates. Surprisingly, an insertion in the CheW homologous domain leads to a more severe defect than an insertion in the CheY homologous domain. The presence of dual pathways for chemotactic signal transduction is consistent with the residual signaling observed in previous studies of cheW mutants (D. W. Hanlon, L. Márques-Magaña, P. B. Carpenter, M. J. Chamberlin, and G. W. Ordal, J. Biol. Chem. 267:12055-12060, 1992).