Co-ordinate expression of genes associated with pathogenicity in Vibrio cholerae requires two transcription activators, ToxR and ToxT. Work carried out to date suggests that ToxR activates transcription of the toxT gene and that ToxT directly activates transcription of several genes whose products play a role in colonization or CT production by V. cholerae. Previous work also suggests that ToxR can directly activate transcription of the CT operon (ctxAB) independently of ToxT, thereby implying a degree of complexity in control of the cixAB operon not found with other genes of the ToxR regulon. We tested the regulatory cascade model of virulence gene expression by constructing strains of classical and El Tor V. cholerae deleted for the coding sequence for the putative DNA-binding domain of toxT. Phenotypic analysis of these strains suggests that V. cholerae has ToxT-dependent and ToxT-independent branches of its virulence regulon. The results also raise questions about the precise role for ToxR in activation of ctxAB transcription.