The genes encoding cholera toxin (CT), ctxAB, are coregulated with those for other Vibrio cholerae virulence factors by a cascade of transcriptional activators, including ToxR, TcpP, and ToxT. Additional regulators that modulate expression of ctxAB during infection were recently identified in a genetic selection. A transposon insertion in vieS, the sensor kinase of the VieSAB three-component signal transduction system, resulted in failure to induce expression of a ctxA-recombinase fusion during murine infection. To determine which components of the VieSAB system are essential for CT regulation, ctxAB transcript levels were assessed by RNase protection assay in various vieSAB in-frame deletion mutants after growth in vitro under virulence gene inducing conditions. A threefold reduction in ctxAB transcript levels was observed for the ΔvieSAB strain; consistent with this, the ΔvieSAB strain produced twofold less CT protein than the wild type, and this defect was complementable in trans. These results suggest that the VieSAB three-component system is required for full activation of the ctxAB operon during in vitro growth as well as during infection. The VieSAB system may regulate ctxAB expression indirectly by affecting production of ToxT, because decreased toxT transcript levels were observed in the ΔvieSAB strain.