A heat-labile cytotoxin was isolated from virulent strains of axenically cultivated Entamoeba histolytica. Strains of E. histolytica representing a spectrum of virulence as determined in animal and in vitro models of disease were examined for cytotoxic activity. Extracts of virulent strain HM1 possessed marked cytotoxic activity, those of moderately virulent strain 200 showed intermediate activity, and those of avirulent strains 303 and Rahman showed no activity. The cytotoxin was partially purified from the cell-free supernatant of sonicated E. histolytica HM1 trophozoites by ammonium sulfate precipitation and gel filtration. Cytotoxic activity was stable in a narrow pH range (6-7.2) and in 1 M NaCl, urea, and guanidine. Specific immune rabbit and human antiserum as well as the protease inhibitors aprotinin, pepstatin, and leupeptin inhibited cytotoxicity. The partially purified cytotoxin did not have any detectable degradative enzymatic activities. Thus, virulent strains of E. histolytica possess an immunogenic cytotoxic protein which may be important in the pathophysiology of amoebiasis.