Two hemolysin genes (ASH3 and ASH4) of Aeromonas salmonicida strain 17-2 and one (ASH1) of A. salmonicida ATCC14174 were cloned into the cosmid vector charomid 9-36 in Escherichia coli DH1. The overall amino acid sequence of the ASH3 was similar to that of the aerolysins of Aeromonas hydrophila and Aeromonas sobria, and hemolysins AHH3, AHH4, and AHH5 of A. hydrophila, and hemolysin ASA1 of A. sobria. The sequence of ASH4 was similar to that of the AHH1 hemolysin of A. hydrophila. The ASH4 hemolysin contains some homologous sequence regions of the Vibrio vulnificus and Vibrio cholerae cytolysin-hemolysin. Both ASH3 and ASH4 DNA probes reacted with all 104 strains of A. salmonicida, whereas the ASH1 DNA probe did not hybridize with any of the 104 strains studied except strain ATCC14174. ASH1 and ASH3 were broad spectrum hemolysins with most activity against rabbit and horse erythrocytes, respectively, whereas ASH4 hemolysin did not lyse bovine and horse erythrocytes. ASH3 and ASH4, but not ASH1, were activated by trypsin.