A clinical isolate (strain 4037) of Kanagawa phenomenon-negative Vibrio parahaemolyticus was studied. Although the strain was judged to be Kanagawa phenomenon-negative by various conventional tests, it produced a new hemolysin (named Vp-TRH, for thermostable direct hemolysin [Vp-TDH]-related hemolysin) that was related to the Vp-TDH produced by ordinary Kanagawa phenomenon-positive V. parahaemolyticus. Vp-TRH was purified by ammonium sulfate fractionation and successive column chromatographies on DEAE-cellulose, hydroxyapatite, and Mono Q. The molecular weight of Vp-TRH was estimated as 48,000 by Sephadex G-100 gel filtration, and the molecular weight of the subunit was estimated to be 23,000 by sodium dodecyl sulfate-slab gel electrophoresis. Thus, like Vp-TDH, Vp-TRH seems to be composed of two subunits. The isoelectric point of Vp-TRH was determined to be 4.6. Vp-TRH showed lytic activities different from those of Vp-TDH on erythrocytes from various animals, especially those from calves, chickens, and sheep. The hemolytic activity of Vp-TRH was labile on heat treatment at 60 degrees C for 10 min, unlike that of Vp-TDH. The immunological similarities, but not the identities of Vp-TRH and Vp-TDH, were demonstrated by Ouchterlony, neutralization, and latex agglutination tests. Thus, we conclude that this clinical isolate of Kanagawa phenomenon-negative V. parahaemolyticus produces a new type of hemolysin that is similar, but not identical, to Vp-TDH, which is usually produced by Kanagawa phenomenon-positive V. parahaemolyticus.