A procedure is described for the isolation of subcellular fractions from epimastigotes of Trypanosoma cruzi. The method could separate most of the nuclei, mitochondria and microsomes. The plasma membranes were purified by discontinuous density gradient centrifugation in alkaline buffer containing sucrose and magnesium. The yield of plasma membrane was 3.7 mg of protein per 10(9) epimastigotes, accounting for approximately 4.2% of the total cell proteins. The plasma membrane obtained from the 34-50% interface of sucrose density gradients was shown, by electron microscopy, to be completely free of other organella and was homogeneous according to enzymatic marker criteria. The specific activity of the 5'-nucleotidase and acid phosphatase were 96- and 5.5-fold, respectively, higher than that in the total cells, suggesting that the enzymes can be considered as good plasma membrane markers for T. cruzi. The results confirmed the possibility of the presence of membrane-bound acid phosphatase of T. cruzi.