Crude lysates of Entamoeba histolytica (strain HM 1:IMSS) analyzed by substrate gel electrophoresis in 12.5% acrylamide separating gels with reducing agents showed six hydrolysis zones with apparent molecular weights of 73,000 (high), 45,000, 36,000 (intermediate), 30,000, 26,000 and 23,000 (low molecular weight proteinases). Amebic lysates fractionated using the procedure of Aley et al. or the procedure of Rosenberg and Gitler and analyzed by the same method show all enzymes in the fractions with the soluble components and only the intermediate and low molecular weight proteinases in the fraction containing internal vesicles or membranes and plasma membrane. Some of these proteinases seem to be integral membrane proteins since they resist treatment with high salt, high urea buffer. All fractions are capable of digesting azocasein. Fractionation of amebic lysates by hydrophobic chromatography using phenyl-Sepharose or phase separation of amebic extracts with Triton X-114 show that proteinases with high, intermediate and low molecular weight behave as hydrophilic proteins while only proteinases of intermediate and low molecular weight behave as hydrophobic proteins. These results suggest that some proteinases are segregated in different compartments of the cell.