The production of cholera toxin (CT) during Vibrio cholerae infection results in the hallmark diarrhea that characterizes the disease cholera. The transmembrane protein ToxR was originally identified as a functional transcriptional activator of ctxAB in a heterologous Escherichia coli system. However, direct ToxR activation of the ctxAB promoter in V. cholerae has not been previously demonstrated. Instead, a regulatory cascade has been defined in which the activators ToxRS and TcpPH modulate ctxAB expression by acting in concert to transcriptionally activate another regulator, ToxT. ToxT, in turn, directly activates ctxAB expression as well as expression of the tcp genes and other virulence-associated genes. In this study, we show that ToxRS directly activates ctxAB in a ToxT-independent manner in a classical biotype V. cholerae, and that this activation requires the presence of bile acids. Although the levels of CT induced by this mechanism are lower than levels induced under other in vitro conditions, the bile-dependent conditions described here are more physiologic, being independent of pH and temperature. We further show that the inability of bile acids to stimulate ToxRS-dependent expression of CT in El Tor biotype strains is related to the differences between classical and El Tor ctxAB promoters, which differ in the number of heptad TTTTGAT repeats in their respective upstream regions. The ability of bile acids to stimulate direct activation of ctxAB by ToxRS depends upon the transmembrane domain of ToxR, which may interact with bile acids in the inner membrane of V. cholerae.