Since the hemolytic activity of extracts from Entamoeba histolytica trophozoites previously described by us might determine at least partially the necrotic lesions of amebiasis, we have continued its characterization in vitro. Using rat erythrocytes as target cells, we have found that cytolysis by E. histolytica trophozoite extracts was (1) dose dependent, (2) localized mainly in a vesicular fraction whose absolute and specific activities were respectively 1.9 and 4.0 times higher than those of total extracts, (3) maximal at pH 8 in the presence of 1 mM Ca++, and (4) progressively lost by heating at 90 degrees C or repeated freezing and thawing. From these results we infer that the major hemolytic factor of E. histolytica may be a protein normally neutralized by an intracellular inhibitor or activated during fractionation.