Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the word. A gene (hlyIII) encoding a hemolysin was cloned and sequenced from V. vulnificus. Nucleotide sequence analysis predicted an open reading frame of 642 bp encoding a 214 amino acid polypeptide that showed 48% sequence identity to the hemolysin III of Bacillus cereus. When HlyIII of V. vulnificus was expressed in Escherichia coli, crude extracts exhibited hemolytic activity similar to that of hemolysin III from Bacillus cereus. A hlyIII isogenic mutant was constructed via insertional inactivation and showed an attenuated virulence compared with the wild-type strain when this mutant was administered intraperitoneally in mice.