The transmembrane DNA-binding protein, ToxR, of Vibrio cholerae is a global transcriptional regulator of virulence gene expression. ToxR has been shown to interact with promoter regions upstream of both the ctxAB operon encoding cholera toxin, and the regulatory gene toxT. Deletion analysis has shown that a repeated sequence, TTTTGAT, is required for ToxR binding and activation of the ctxAB promoter. However, this sequence is not found upstream of the toxT promoter. Genetic selections using P22 challenge phages were used to define sites within the promoter for ctxAB which are critical for ToxR-DNA interactions. Single-base-pair changes and deletion mutations that impair ToxR binding cluster within two regions: -57 to -69 within two of three tandem TTTTGAT sequences; and from -39 to -47, between the repeat sequences; and the -35 region of the promoter. ToxR does not bind to a synthetic target that has three tandem repeats which lack a flanking upstream and downstream sequence. These results suggest that the ToxR-binding site lies immediately upstream of the -35 position of the ctx promoter, and that the affinity of ToxR binding to this site is influenced by the repeat sequences.