In bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes, relA, spoT, and relV, govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogen Vibrio cholerae. Here we report functional characterization of the dksA gene of V. cholerae (dksA Vc ), coding for the protein DksA Vc . Extensive genetic analyses of the ⌬dksA Vc mutants suggest that DksA Vc is an important component involved in the stringent response in V. cholerae. Further analysis of mutants revealed that DksA Vc positively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), under in vitro conditions. We found that DksA Vc upregulates expression of the sigma factor FliA ( 28 ), a critical regulator of motility in V. cholerae. Altogether, it appears that apart from stringent-response regulation, DksA Vc also has important roles in fine regulation of virulence-related phenotypes of V. cholerae.