Glycosylphosphatidylinositol (GPI)-anchored surface proteins of Leishmania seem to be responsible for the resistance of these parasites to serum complement-mediated cytolysis, uptake of promastigotes by macrophages and protection from degradation in the phagolysosome. The main objective of the present study was to obtain GPI-anchored proteins of L. amazonensis and to reconstitute them into liposomes. Since the treatment of the membrane of Leishmania with glycosylphosphatidylinositol-specific phospholipase C (PI-PLC) removes the ability of these molecules to re-incorporate into liposomes, we standardized a rapid and efficient method by using detergent treatment to obtain proteins with intact GPI anchor. Detergents are usually used for the isolation and purification of membrane proteins. Triton X-114 phase partitioning has frequently been used to obtain preparations enriched with GPI-anchored proteins and other hydrophobic proteins from crude cellular homogenates. Membrane fractions of L. amazonensis promastigotes were solubilized (0.5 mg/ml of total protein) in 1% Triton X-114 at 4oC. After phase separation at 20oC, the detergent rich phase was treated (0.2 mg/ml of total protein) with 0.2 U/ml of PI-PLC of the Bacillus thuringiensis. Aliquot of all steps obtained during the solubilization process by detergent (detergent-rich phase, aqueous phase and membrane-containing pellet) and also the resuspended pellet and supernatants obtained after treatment with PI-PLC were analyzed by SDS-PAGE and Western blotting. The membrane fraction of parasite showed three major bands with approximately 65-60, 52 and 50-47kDa when stained for protein that revealed high intensity antiserum against total L. amazonensis antigenic determinants. Similar results were observed among the samples of the detergent-rich phase and the sample treated with PI-PLC, but the electrophoresis analysis of the samples treated with PI-PLC showed a diminution of the molecular mass of these three major bands. These results suggest loss of their covalently bound lipid moiety, indicating that these proteins were anchored to the membrane domains through the GPI anchor. The standardized method using Triton X-114 could be used for the reconstitution of antigenic GPI-proteins into liposomes for use as potential adjuvant activity in vaccines against several protozoan organisms.  Supported by: CNPq, CAPES, FAPESP.