Vibrio vulnificus is an estuarine bacterium that opportunistically infects humans with underlying hepatic diseases, heavy alcohol drinking habits, and other immunocompromised conditions. A locus in the V. vulnificus genome was cloned and sequenced, which showed similarities to the bacterial two-component signal transduction system. The locus encoded a putative sensor kinase, designated vvgS, and a divergently transcribed putative response regulator, designated vvgR. VvgS was predicted to be a protein belonging to the tripartite hybrid sensor kinase subfamily such as ArcB and BvgS. VvgR showed similarities to well known response regulators. An insertional mutation of vvgS in a V. vulnificus strain led to a remarkable decrease of cytotoxicity to HeLa cells, while the production of exotoxins, the hemolysin and the protease, slightly increased by the vvgS mutation. Adhesion to HeLa cells only slightly decreased. However, the vvgS mutation had no effect on the motility of V. vulnificus. The vvgS mutation resulted in a significant decrease of lethality to mice. These results indicate that vvgRS two-component system plays a very important role in regulating novel virulence factor(s) of V. vulnificus associated with cytotoxicity other than hemolysin and protease during the infection process.