Trypanosoma equiperdum and Trypanosoma evansi were purified by three or four cycles of low-speed centrifugation and final filtration through DEAE cellulose. The purified trypanosomes were used in comparative biochemical and immunological studies. Comparative polypeptide pattern analysis revealed that T. equiperdum showed 21 polypeptide bands, whose Mr ranged from >200 to 14.8 kDa. T. evansi showed 25 polypeptide bands in the Mr range 97-14.8 kDa. The main differences were associated with the presence of secondary bands, relative intensity and the number of bands. Both species gave seven glycoprotein bands; those of 97 and 68 kDa were present in T. equiperdum but absent in T. evansi. Bands of 61 and 28 kDa were present in T. evansi but not in T. equiperdum. Anti-T. equiperdum sera recognized four homologous antigens and cross-reacted with three antigens of T. evansi. Anti-T. evansi sera recognized three homologous antigens and cross-reacted with four T. equiperdum antigens. Four identical proteolytic protease bands were present for both species, while only one surface protein was detected for each species: 66 kDa for T. equiperdum and 62 kDa for T. evansi.