Leukotoxic activity was assayed in clinical isolates of Enterobacter cloacae. Two strains were selected out of 38 by their greater hemolytic activity in blood agar plates. Leukotoxin was purified by salt precipitation, dialysis, chromatography by gel filtration, and high pressure liquid chromatography (HPLC). Human leukocytes, when incubated with purified E. cloacae toxin, showed high percentages of death and lysis, with time and dose dependence. The chromatographic profile of gel filtration presented three protein peaks and toxic activity was detected in the second peak. After HPLC, leukotoxin coeluted with the hemolytic activity and both activities were detected only after 2-mercaptoethanol treatment. Coomassie-stained sodium dodecyl sulfate--polyacrylamide gels showed a single band. This band was estimated to represent a protein of 13300 Da on the basis of both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography.