Specialized membrane microdomains enriched in sphingolipids, cholesterol and specific subsets of proteins are present in eukaryotic plasma membranes, and also have been reported in Leishmania and Trypanosoma brucei. Usually these domains are detergent resistant membranes (DRMs) and can be isolated by density gradient ultracentrifugation on the basis of their low density and insolubility in cold non-ionic detergent (Triton X-100 or BRIJ 98). In this work, we analyzed the presence of glycosylinositol phosphorylceramides (GIPCs), the major glycoconjugate present in Trypanosoma cruzi epimastigotes, in DRMs. Epimastigote forms of T. cruzi (strains CL and G) were grown in LIT medium. Triton X-100 soluble and insoluble parasite components at 4˚C, were separated by centrifugation at 14.500 x g, and their lipids were extracted with mixture of isopropanol/hexane/water (55:20:25,v/v/v). High performance thin layer chromatography (HPTLC) analysis showed that about 88% of total GIPCs and 58% of total inositol phosphorylceramide (IPC) are present in detergent-insoluble fraction obtained after treatment of parasites with Triton X-100 1% at 4˚C. On the other hand, most of epimastigote phospholipids were solubilyzed by this detergent at 4˚C. By Western blotting it was also observed presence of four components in the detergent-insoluble fraction (80, 48, 38, 32kDa) which are recognized by sera from patients with Chagas Disease. In order to confirm that the 4˚C detergent-insoluble fraction described above represent DRMs, membrane microdomains of low density was obtained by ultracentrifugation in a sucrose gradient and tested with mAb BST-1 (anti T. cruzi GIPCs) by HPTLC immunostaining and Western blotting. Presence of GIPCs were detected mainly in fraction 3 of sucrose gradient, which corresponds to the fraction containing low density membranes. These results show the presence of specialized detergent-resistant membranes in T. cruzi epimastigotes, which are enriched in GIPCs, IPCs and specific glycoproteins.