Aeromonas spp. have been isolated from drinking water, lakes and a variety of foods. The virulent Aeromonas spp. have been associated with several categories of human infections, such as gastroenteritis, meningitis, septicemia, and wound infections by producing exotoxins. The structural gene for the hemolytic toxin aerolysin from a transposon-derived Aeromonas trota strain AK253 has been cloned into the plasmid vector pGEM5Z(-) with selection of kanamycin resistance. The resultant clone, pAK71 demonstrated aerolysin activity, indicating that the genes for this enzyme was adjacent to the transposon insertion. The 5-kb EcoRI fragment encoding the entire operon of hemolysin was subcloned from pAK71 into pGEM3Z. E. coli JM109 containing recombinant plasmid pAK72 exhibited zones of hemolysis after 16 h incubation on blood agar plates at 37°C. We were unable to find extracellular hemolytic activity in culture supernatant of E. coli harboring pAK72. However when concentrated (20X) the culture supernatant from E. coli JM109(pAK72) was able to show hemolytic activity on sheep blood. The low level of extracellular hemolysin may be due to the absence of secretion mechanism in E. coli.