Clarified homogenates prepared from different strains of axenically reared Entamoeba histolytica were assayed for acid and neutral proteinase activity, cytotoxicity, and protein content. The homogenates of the more virulent strains contained more acid or neutral proteinase and more toxin units per 10(6) trophozoites. Toxicity and neutral proteinase activities had a similar pattern of distribution. The cytotoxicity of the gel-filtered proteins of the studied strains followed the same rank order as their reported virulence. The more virulent strains (HM-1 and Rahman) had significantly more neutral proteinase activity than less virulent strains (HK-9, Laredo and Huff). Purified extracts of HM-1 and Rahman strains contained more toxin per milligram of protein and exhibited a lower cytotoxicity end point than those of the less virulent strains. The most cytotoxic fraction of each strain had a molecular weight of approximately 24,000, and all cytotoxins were antigenically similar. A more virulent strain (HM-1) secreted more cytotoxic activity when incubated under tissue culture conditions than did a less virulent strain (HK-9). Cytotoxic material secreted by strain HM-1 during axenic growth had the same molecular weight as that isolated from trophozoites. The Entamoeba histolytica toxin studied may be a neutral proteinase that is present in greater quantities in more virulent strains, thus accounting for their greater cytotoxicity in vitro and invasiveness in vivo.