The Kanagawa phenomenon induced by the thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus is almost exclusively associated with clinical strains, and TDH has been considered an important virulence factor. However, Kanagawa phenomenon-negative strains isolated from patients with diarrhea have recently been shown to produce TDH-related hemolysin (TRH). We studied the distribution of the tdh gene encoding TDH and the trh gene encoding TRH in vibrios by hybridization analyses. The presence or absence of the tdh gene and the trh gene in 285 strains of V. parahaemolyticus was examined by the DNA colony hybridization test with a tdh gene-specific probe and a newly constructed trh gene-specific probe. For assessment of the importance of TRH, many Kanagawa phenomenon-negative clinical strains (35.4% of all strains) were included. Of 214 clinical strains of V. parahaemolyticus, 112 strains (52.3%) had the tdh gene only, 52 strains (24.3%) had the trh gene only, and 24 strains (11.2%) carried both the tdh and the trh gene. The coexistence of the tdh and trh genes in these 24 strains was confirmed by Southern blot hybridization analysis. Of 71 environmental strains, 5 strains (7.0%) hybridized very weakly with the trh gene probe and none hybridized with the tdh gene probe. These results suggest that TRH as well as TDH is an important virulence factor of V. parahaemolyticus. Among 118 strains of other Vibrio species examined for the trh gene, only 1 strain of Vibrio furnissii gave a very weak hybridization signal. Among 48 representative trh gene-positive strains of V. parahaemolyticus, only 18 strains (37.5%) were found to produce TRH in culture medium when examined by a sensitive enzyme-linked immunosorbent assay method.