Vibrio mimicus (V mimicus), a causative agent of gastroenteritis and food poisoning, secretes a 63-kDa enterotoxic hemolysin as the most potent virulence factor. The vmhA gene encoding an 83-kDa precursor of the hemolysin was expressed from the early to late log phase of the bacterial growth, and the 79-kDa inactive protoxin was detected from the culture supernatant in the same growth phase. The N-terminal amino acid sequence of the protoxin was determined to be NH2-Asn-Ile-Ser-Asp-Pro-Val indicating cleavage of the Ala(25)-Asn(26) bond by a signal peptidase. In contrast, the hemolytic activity and the mature hemolysin in the culture supernatant were detected only at the late log phase. The maturation of the hemolysin, therefore, is suggested to be achieved by two-step processing, cleavage of the signal peptide followed by the growth phase-dependent removal of the 16-kDa propeptide.