The molecular weights of the major polypeptides of Entamoeba histolytica NIH 200 soluble whole extract, and its five fractions eluted from a Sephadex G-200 column, were in the range 150,000–9,000 daltons according to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of SDS-PAGE analysis of 125I surface-labeled amebae revealed more than 12 bands, eight of which corresponded to those recovered in soluble extract. Considering the pronounced phagocytosis and the rapid cell membrane turnover of the ameba, it seems likely that the same antigenic components would be present both on and within the ameba at different stages. The first four gel-filtered fractions were immunogenic and showed non-identical precipitates with sera from 12 Swedish and 12 Ethiopian patients with verified amebic disease. Seven polypeptides in the whole extract reacted with human anti-ameba IgG covalently bound to CNBr activated Sepharose 4B. Immune replica (Western blotting) of SDS-PAGE of whole extract produced by human anti-ameba antisera demonstrated eight bands. The response to the different amebic antigens was not correlated to clinical symptoms, duration of illness or origin of ameba strain. Thus the present results did not support the use of purified fractions rather than the whole soluble extract for diagnostic tests.