The tdh gene of Vibrio parahaemolyticus which encodes the thermostable direct hemolysin has been found in some strains of other Vibrio species. Analysis of seven tdh genes cloned from V. parahaemolyticus, Vibrio mimicus, and non-Ol Vibrio cholerae revealed that all tdh genes were flanked by insertion sequence-like elements (collectively named ISVs) or related sequences derived from genetic rearrangement of ISVs. The ISVs possessed 18-bp terminal inverted repeats highly homologous to those of IS903 (2-to 4-bp mismatch) and were 881 to 1,058 bp long with <33.6% sequence divergence. These features and nucleotide sequence similarities among ISVs and IS903 (overall homologies between ISVs and IS903, ca. 50%) strongly suggest that they were derived from a common ancestral sequence. A family of ISVs were widely distributed in Vibrio species, often regardless of the possession of the tdh genes, and one to several copies of the ISVs per organism were detected. A strain of V. mimicus possessed two copies of the ISVs flanking the tdh gene and three copies unrelated to the tdh gene. However, the transposition activity of the ISVs could not be demonstrated, probably because they had suffered from base changes and insertions and deletions within the transposase gene. The possible mode of ISV-mediated spread of the tdh gene is discussed from an evolutionary standpoint.Vibrios are widely distributed in the marine environment, and some species can cause gastroenteritis in humans through seafood consumption (5, 18). The pathogenic mechanism of one such bacterium, Vibrio parahaemolyticus, is not clearly understood, but the thermostable direct hemolysin (TDH) produced by this organism is considered an important virulence factor (37). Recently, some strains belonging to other Vibrio species isolated from diarrheal patients were found to produce hemolysins similar to TDH. These species include non-O1 Vibrio cholerae (44, 45), Vibrio mimicus (15), and Vibrio hollisae (46).We have been studying the gene (tdh) encoding TDH. Five tdh genes cloned from V. parahaemolyticus (designated tdhl to tdh5) have been sequenced, and it was found that the coding regions had a sequence divergence of less than 3.3% (2,29,30). The genes encoding the TDH-like hemolysins of other Vibrio species have also been cloned (16, 31). The coding sequences of the genes cloned from non-O1 V. cholerae, V. mimicus, and V. hollisae had 96.7% or more, 97.0% or more, and 93.3% or more homologies, respectively, with genes tdhl to tdh5. Therefore, these genes were included in the tdh gene family and were named NAG-tdh, Vm-tdh, and Vh-tdh, respectively (1, 39, 43).While the majority of clinical strains of V. parahaemolyticus and V. hollisae possessed the tdh gene (28), only some clinical strains of non-O1 V. cholerae and V. mimicus had the tdh gene (16,31). Evidence to support the hypothesis of plasmid-mediated transfer of the tdh gene between Vibrio species was obtained by comparative analysis of two plasmid-borne tdh genes (tdh4 and NAG-tdh) (1), and it could explain the ...