The extracellular hemolysin (AHH1) gene of Aeromonas hydrophila ATCC7966 was cloned into Charomid9-28 in Escherichia coli DH1, and its complete nucleotide sequence determined. Escherichia coli carrying this gene expressed an extracellular heat-labile hemolysin for rabbit red blood cells. The minimum size of the coding region of the 2.6 kilobase-pair BamHI-SphI fragment was subcloned into pUC118 and pUC119, selecting for hemolytic activity. The nucleotide sequence of this region contained a single open reading frame of 1734 base pairs, corresponding to a protein of 577 amino acid residues (63,658 daltons). A consensus promoter sequence was present upstream of the AHH1 open reading frame. Maxicell analysis of [35S]methionine-labelled proteins in E. coli CSR603 carrying the AHH1 plasmid suggested that AHH1 gene codes for an approximately 60,000 dalton polypeptide. By colony DNA-DNA hybridization analysis, the AHH1 gene was detected in 43 of 62 hemolysin-producing strains of A. hydrophila (isolated from various sources and areas) and in all 43 hemolysin-producing strains of A. salmonicida (isolated from fish). Three hemolysin-negative strains of A. hydrophila did not react with the AHH1 probe, whereas three non-hemolytic A. salmonicida strains hybridized with the probe.