Co-ordinate expression of many virulence genes in Vibrio cholerae is under the control of the ToxR and ToxT proteins. These proteins function in a regulatory cascade in which ToxR is required to activate toxT, and ToxT activates virulence genes. The precise mechanism for ToxR activation of toxT is unknown, but data presented in this report suggest a direct involvement of ToxR. Primer extension and gene fusion analyses identified a ToxR-regulated promoter directly upstream of toxT, immediately following a region of inverted repeats capable of terminating transcription. Gel mobility shift studies indicate that ToxR binds DNA within the inverted repeat region, yet preliminary evidence suggests that ToxR binding alone is not sufficient for activation of toxT. Possible mechanisms of ToxR-dependent toxT expression are discussed.