The lipopeptidophosphoglycan from Trypanosoma cruzi is a glycosylated inositol-phosphoceramide isolated from epimastigotes at the stationary phase of growth (4-5 days). We have now purified two similar glycoinositolphospholipids (glycoinositolphospholipid A and glycoinositolphospholipid B) from epimastigotes after the second day of culture growth. [3H]Palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in glycoinositolphospholipid A and into ceramide in glycoinositolphospholipid B. The lipids were released by incubation with glycosylphosphatidylinositol-specific phospholipase C from Bacillus thuringiensis or by chemical methods. After alkaline hydrolysis, the lipids were analysed by GLC/MS. In glycoinositolphospholipid A the resulting lipids corresponded to 1-O-hexadecylglycerol and palmitic acid. The ceramide components in glycoinositolphospholipid B are sphinganine, palmitic acid and lignoceric acid. The oligosaccharides could be degraded by nitrous acid and further enzymic treatment showed that the two glycoinositolphospholipids isolated from T. cruzi share the common core structure of the glycosylphosphatidylinositol membrane anchors. The microheterogeneity was determined, as well as the substitution by galactose, and was mainly in the furanose configuration as was previously described for lipopeptidophosphoglycan. However, methylation analysis indicated that 20% of the galactose is in the pyranose form. Both glycoinositolphospholipids mainly differ in the lipid moiety.