Four tdh genes encoding thermostable direct hemolysin (TDH) cloned from two representative strains of V. parahaemolyticus (tdh 1 and tdh 2 from a hemolytic strain, tdh3 and tdh4 from a non-hemolytic strain) have different nucleotide sequences (the maximum divergence: 3.3%). In this study, each tdh gene product was purified from the lysate of Escherichia coli cells carrying the cloned gene and their properties were compared to investigate the influence of the amino acid substitutions caused by these base changes. The four tdh gene products showed different electrophoretic mobilities under non-denaturing conditions. All the gene products had hemolytic activities for various animal erythrocytes, stimulated vascular permeability in the rabbit skin, and were lethal to mice, although their potencies were slightly different. Antigenicities of the four gene products were indistinguishable. These results indicate that the four tdh genes have evolved to maintain a fundamental molecular structure and biological activities of the gene products and minor structural and/or charge differences of the molecules are perhaps responsible for the slight divergence of their biological activities.