Axenic amoebic antigen was chromatographed on Sephadex G-200. Two peaks divided into three fractions were obtained which were concentrated by lyophilization. Guinea-pigs were given a series of subcutaneous injections of crude Entamoeba histolytica antigen and its chromatographic fractions mixed with Freund's complete adjuvant. After three immunizing doses, the animals were challenged with intracaecal injection of a virulent isolate of amoebae (Strain No. H-25) and were killed seven days later. The antibody response was studied by indirect haemagglutination (IHA) and counter-current immunoelectrophoresis (CIEP) before intracaecal inoculation and at the time of killing. Fraction I, which had the highest molecular weight, stimulated high levels of antibodies in guinea-pigs and showed 92% protection. Only one animal of 12 inoculated had gross lesions and these were very mild. The caecal contents of three of the 11 animals without gross lesions were positive for amoebae but no tissue invasion was observed. Partial protection was observed with other antigens--30% with whole amoebae, 18% with Fraction II and 30% with Fraction III. In most there were multiple ulcers of the deep penetrating type and amoebae either penetrating and invading the muscular layer or the lytic process extending up to the muscular layer. In one animal, immunized with whole amoebic antigen, multiple hepatic lesions were also observed and trophozoites of E. histolytica were demonstrated histologically.