ToxT, a member of the AraC family of transcriptional regulators, controls the expression of several virulence factors in Vibrio cholerae. In the classical biotype of V. cholerae, expression of toxT is regulated by the same environmental conditions that control expression of the virulence determinants cholera toxin and the toxin coregulated pilus. Several genes that activate toxT expression have been identified. To identify genes that repress toxT expression in nonpermissive environmental conditions, a genetic screen was used to isolate mutations which alter the expression of a toxT-gusA transcriptional fusion. Several mutants were isolated, and the mutants could be divided into two classes. One class of mutants exhibited higher expression levels of toxT-gusA at both the nonpermissive pH and temperature, while the second class showed elevated toxT-gusA expression only at the nonpermissive pH. One mutant from the second class was chosen for further characterization. This mutant was found to carry a TnphoA insertion in a homolog of the Escherichia coli pepA gene. Disruption of pepA in V. cholerae resulted in elevated levels of expression of cholera toxin, tcpA, toxT, and tcpP at the noninducing pH but not at the noninducing temperature. Elevated levels of expression of toxT and tcpP at the nonpermissive pH in the pepA mutant were abolished in tcpP toxR mutant and aphB mutant backgrounds, respectively. A putative binding site for PepA was identified in the tcpPH-tcpI intergenic region, suggesting that PepA may act at the level of tcpPH transcription. Disruption of pepA caused only partial deregulation at the noninducing pH, suggesting the involvement of additional factors in the pH regulation of virulence genes in V. cholerae.