Thermostable direct haemolysin of Vibrio parahaemolyticus has been shown to be a major virulence factor. The Kanagawa phenomenon (KP), haemolysis induced by this haemolysin on a special blood agar medium, is strongly associated with clinical strains. We have been studying the expressions of various tdh genes encoding this haemolysin to elucidate the significance of the tdh genes possessed by KP-negative strains isolated from patients. We examined the importance of the promoter sequence variation for expression level of the tdh gene in this study. Only the tdh2 gene, one of the two tdh genes (tdh1 and tdh2) present in a KP-positive strain, was previously shown to be responsible for the haemolytic activity of the KP-positive strain. The tdh1- and tdh2-lacZ fusions were used to determine and analyse the promoter sequence by primer extension and site-directed mutagenesis methods. Two bases (positions −24 and −34) within the determined tdh2 promoter sequence were shown to be mostly responsible for the difference in the promoter strength between the tdh2 and tdh1 genes both in Escherichia coli and in V. parahaemolyticus backgrounds. Representative tdh promoters of KP-negative strains are close to the tdh2 promoter; they differ at position −34 but have the same base at position −24 as the tdh2 promoter. We demonstrated that base substitution of the tdh promoters of KP-negative strains only at position −34 is sufficient to increase the expression of these genes to the KP-positive level. Therefore, the tdh genes of KP-negative strains are considered to be potentially important because they can generate a KP-positive subclone by a point mutation in their promoters.