Virulence factor expression in Vibrio cholerae is controlled by the transcriptional regulatory protein ToxT. ToxT activates transcription of the genes encoding cholera toxin (ctx) and the toxin co-regulated pilus (tcp), as well as accessory colonization factor (acf) genes. Previous studies of ToxT, a member of the AraC family of proteins, have revealed that it consists of two domains, an N-terminal dimerization and environmental sensing domain, and a C-terminal DNA binding domain. In this study, comprehensive scanning alanine mutagenesis was utilized to identify amino acids critical for the function of ToxT. Forty-eight proteins with Ala substitutions (of 267 total) exhibited defects in ToxT-dependent activation (>90% reduction) in both a V. cholerae acfA-phoA reporter strain and a Salmonella typhimurium ctxAp-lacZ reporter strain. Most of these mutant proteins also caused reductions in cholera toxin (CT) and toxin coregulated pilus (TCP) expression in a DeltatoxT V cholerae strain under in vitro virulence factor inducing conditions. Further analysis with a LexA-based reporter system revealed that one of the 20 Ala substitutions in the N terminus (F151A) diminishes dimerization, and this residue is located in a region of predicted alpha-helical structure, thus identifying a putative dimer interface. Ala substitutions in two putative helix-turn-helix (HTH) recognition helices that caused differential promoter activation (K203A and S249A) did not appear to alter specific DNA binding, suggesting these residues contribute to other aspects of transcriptional activation. A number of Ala substitutions were also found that result in a higher level of ToxT transcriptional activity, and these mutations were almost exclusively found within the N terminus, consistent with this domain being involved in modulation of ToxT activity. This study illuminates the contribution of specific amino acids to the dimerization, DNA binding, and transcriptional activity of ToxT.