Hemolyin/cytolysin (VvhA) is one of the representative exotoxins produced by Vibrio vulnificus. Cytotoxic mechanism of VvhA has been extensively studied. However, there have been controversies concerning the pathogenic significance since vvhA(-) mutant showed no LD50 change in mice. In this study, we investigated whether VvhA is expressed in vivo. When V. vulnificus was cultured in the presence of normal pooled human serum, substantial amount of VvhA was detected by ELISA and the transcription of vvhA was also evidently confirmed by RT-PCR and a transcriptional reporter assay. To investigate whether VvhA is expressed in vivo, mice were infected with V. vulnificus and bacterial RNAs were harvested from the mice. In vivo vvhA transcription was observed evidently by RT-PCR. We hereby propose that VvhA is substantially produced in vivo and would contribute to the pathogenesis of V. vulnificus septicemia. (C) 2004 Elsevier Inc. All rights reserved.