The soluble antigens of Entamoeba histolytica trophozoites were analysed in detail by biochemical and immunochemical methods. The antigen was highly complex and heterogeneous as revealed by Sephacryl S-300 column chromatography, which showed four distinct fractions. The molecular mass of fractions FI, FII, FIII and FIV was 660, 170, 65 and 13 kDa, respectively. Protein was the major constituent in crude soluble antigen (CSA) and fractions FI and FII (67%, 80% and 90%, respectively). Polysaccharide was predominant in the FIII fraction (59%). Antigenic activity observed after different physico-chemical treatments revealed that CSA and FI antigens were predominantly glycoprotein in nature. However, the antigenicity of FIII antigen was greatly reduced after sodium meta-periodate treatment, whereas no alteration in reactivity was discerned after trypsin treatment. Sodium dodecyl sulphate-polyacryl-amide gel electrophoresis (SDS-PAGE) analysis demonstrated nearly 28 Coomassie blue bands for CSA and 20, 16, 15 and 3 polypeptide bands for the FI, FII, FIII and FIV fractions, respectively. The molecular mass of the polypeptides of these bands ranged from 210 to 20 kDa. Antigenic activity was observed in CSA and in the first three fractions, both in counter immunoelectrophoresis (CIEP) and in enzyme-linked immunosorbent assay (ELISA). However, the highest antigenic activity was noted in fraction FI. Major immunoreactive polypeptides of CSA and FI antigens against whole trophozoite antibody were observed in the 10- to 170-kDa regions. However, major differences in the immunoreactivity of the two antigens were noted at 116 and 14 kDa for FI antigen and at 84, 30 and 20 kDa for CSA.(ABSTRACT TRUNCATED AT 250 WORDS)