We have cloned and sequenced the gene encoding thermostable direct hemolysin (TDH), a possible virulence factor in Vibrio parahaemolyticus gastroenteritis, from a Kanagawa-phenomenon-positive strain, T4750. This strain was found to contain two sequences (tdhA and tdhS) homologous to the tdh gene previously reported by Nishibuchi and Kaper [J. Bacteriol 162 (1985) 558-564] and Taniguchi et al. [Microb. Pathog. 1 (1986) 425-432]. Sequence homology of the coding regior between tdhA and tdhS was 97.2%. The deduced amino acid (aa) sequence of TdhA, excluding the putative signal peptide was identical to that of TDH protein purified from V. parahaemolyticus [Tsunasawa et al., J. Biochem. 101 (1987) 111-121] except for Glu118 instead of Gln118. Although the aa sequence deduced from the second gene, tdhS, differed in eight residues from the TDH protein, it agreed with the sequence of Tdh deduced from the previously cloned tdh gene. Both tdhA and tdhS expressed biologically active hemolysins in Escherichia coli. While the apparent molecular size of TDH purified from a culture supernatant of V. parahaemolyticus T4750 was identical to TdhA protein synthesized in E. coli, it was larger than TdhS. Only one band was detected in the culture supernatant of V. parahaemolyticus T4750 by Western blotting; its mobility was indistinguishable from that of purified TDH. These data suggest that tdhA is the structural gene for TDH found in the culture supernatant of V. parahaemolyticus T4750, and that there was only partial, if any, tdhS expression in the strain T4750 under the test conditions employed.