In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRSVc) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRSVp), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1.2-kb HindIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxRVv, toxSVv, and htpGVv were cloned. ToxRVv shared 55.0 and 63.0% sequence homology with ToxRVc and ToxRVp, respectively. ToxSVv was 71.5 and 65.7% homologous to ToxSVc and ToxSVp, respectively. The amino acid sequences of ToxRSVv showed transmembrane and activity domains similar to those observed in ToxRSVc and ToxRSVp. Western blot analysis proved the expression of ToxRVv in V. vulnificus. ToxRSVv enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene (vvhA) fivefold. ToxRSVv also activated the ToxRVc-regulated ctx promoter incorporated into an E. coli chromosome. A toxRVv null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxRVv mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxRVv may regulate the virulence expression of V. vulnificus.