The relationship between phenotypic variation and nucleotide sequence variation of the gene encoding Vibrio parahaemolyticus thermostable direct haemolysin (tdh gene) was examined. Strains showing a typical haemolysin-positive phenotype carried two chromosomal gene copies (designated tdh1 and tdh2) while tdh-gene-positive strains showing a weakly positive or negative haemolysin phenotype possessed only a single chromosomal gene copy. Both gene copies from a typical haemolysin-positive strain were cloned and sequenced and possessed 97.2% homology. Comparison of the amino acid sequence predicted from the nucleotide sequence with the protein sequence determined by Edman degradation as well as construction of a tdh1-deficient yet haemolytic strain of V. parahaemolyticus suggest that the tdh2 locus is primarily responsible for the haemolytic phenotype. Two other tdh gene copies were cloned from a phenotypically negative strain which was unusual in that it contained one gene copy on a plasmid (designated tdh4) in addition to a single copy on the chromosome (tdh3). Both tdh3 and tdh4 were expressed in Escherichia coli and TDHs with haemolytic activity were produced. These gene copies were sequenced and shared 96.7% homology with the tdh1 gene. The V. parahaemolyticus strain carrying tdh3 and tdh4 gene copies did not produce detectable amount of tdh-specific RNA transcript. It seems, therefore, that differences in the transcriptional control are primarily responsible for the differences seen in haemolytic phenotype.