Vibrio parahaemolyticus, one of the human-pathogenic vibrios, is a Gram-negative halophilic bacterium that naturally inhabits marine and estuarine environments. The bacterium causes food-borne gastroenteritis, most frequently associated with the consumption of raw or undercooked seafood (1). Consumption of sufficiently high numbers of the organism can cause watery diarrhea, septicemia and even death (7,10). Almost all the clinical isolates of V. parahaemolyticus show β-hemolysis on Wagatsuma agar, a special blood agar medium (24). The hemolysis has been called the Kanagawa-phenomenon (KP), and is considered a good marker to differentiate human pathogenic V. parahaemolyticus from the non-pathogenic strains. To date, the thermostable direct hemolysin (TDH), has been shown to have a role in KP (6,27). In 1988, Honda et al. (9) discovered a new hemolysin in KP-negative clinical V. parahaemolyticus isolates. This hemolysin was referred to as TDH-related hemolysin (TRH). It is immunologically similar to TDH, and shares approximately 67% identity with TDH at the amino acid level (9). TDH and TRH show several common biological properties, such as hemolytic activity, enterotoxicity and cytotoxicity (5,6,8,10,16,17,23,25,29,31). V. parahaemolyticus strains isolated from patients with diarrhea produce TDH or TRH or both, whereas environmental isolates rarely produce these proteins (6, 27). Thus, TDH and TRH have been considered to be involved in the gastrointestinal disorders caused by V. parahaemolyticus (6).Although live V. parahaemolyticus shows cytotoxicity and enterotoxicity (2, 15, 25), the virulence factors responsible for these activities have not been fully elucidated. Recently, we completed the genome sequencing of a KP-positive (TDH-producing) V. parahaemolyticus RIMD2210633 strain (13). In this study, we constructed tdh-deletion mutants from the sequenced RIMD2210633 strain, and examined the hemolytic activity, cytotoxicity and enterotoxicity of the mutant strains Abstract: The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of Vibrio parahaemolyticus. We have recently completed the genome sequence of a TDH-producing V. parahaemolyticus strain, RIMD2210633. In this study, we constructed tdh-deletion mutants from the sequenced strain by homologous recombination and analyzed their phenotypes. Although the deletion of both copies of tdh completely abolished the hemolytic activity of the wild-type strain, the deletion did not affect the cytotoxicity to HeLa cells. Enterotoxicity, assayed by the rabbit ileal loop test, was lowered by tdh deletion, but the mutant still showed partial fluid accumulation in rabbit intestine. These results indicate that the cytotoxicity and enterotoxicity of TDH-producing V. parahaemolyticus are not explained by TDH alone, and suggest that an unknown virulence factor(s) could be involved in these pathogenic activities.