The structural gene and regulatory element for a cytolytic enterotoxin of a diarrheal isolate, SSU, ofAeromonas hydrophilawas cloned and its DNA sequence was determined. A complementary, mixed synthetic oligonucleotide based on the first 10 NH2-terminal amino acid residues of theAeromonascytolytic enterotoxin was used as a probe to screen a genomic library constructed in bacteriophage EMBL3. Cell lysates ofEscherichia coli(λCH4), containing the cytolytic enterotoxin gene, lysed rabbit red blood cells and destroyed Chinese hamster ovary cells, caused fluid secretion in rat ileal loops, and were lethal to mice when injected intravenously. All biological activities associated with the cytolytic enterotoxin were neutralized by rabbit homologous polyclonal antibodies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent Western blot analysis of the cell lysate ofE.coli(λCH4) revealed a protein band of approximately 52 kDa, using antisera to the cytolytic enterotoxin or antibodies generated against a synthetic peptide to the toxin. DNA sequence analysis of a 2.8-kbSalI-BamHI fragment revealed the presence of one large open reading frame (1479 bp) that would encode a protein of 54.5 kDa, a precursor form of the cytolytic enterotoxin, with a 23 amino acid leader peptide. Despite a significant amount of homology at the DNA and amino acid levels between our cytolytic enterotoxin and two aerolysins ofAeromonasspecies, variation in the restriction maps of these three toxin genes was prominent. Likewise, considerable divergence in DNA sequence was observed upstream of the structural genes for the reported aerolysins and our cytolytic enterotoxin, suggesting that these structurally similar toxin molecules may be regulated differently. Finally, our data showed that the cytolytic enterotoxin from a diarrheal isolate, SSU, ofA.hydrophilaexhibited characteristics that were unique compared with those of the reported aerolysins.Key words:Aeromonas hydrophila, cytolytic enterotoxin, aerolysin, cholera toxin.