The glycolipids of the protozoan Leishmania major strain LRC-L119 belong to a class of glycoinositol phospholipids (GIPL) that show partial structural homology to the phosphatidylinositol-containing glycolipid membrane anchors of several eukaryotic proteins and the lipid moiety of L. major lipophosphoglycan. The GIPLs were the only glycolipids detected and were purified by octyl-Sepharose and thin layer chromatographies. Analysis of the native and dephosphorylated glycolipids (GIPLs 1-6) by gas chromatography-mass spectrometry revealed that the glycan moieties have between 4 and 10 saccharide residues and all contain mannose, galactose, and non-N-acetylated glucosamine. Some of the GIPLs also contain glucose (GIPL-6) and hexose monophosphate residues (GIPL 4-6). The presence of an inositol phospholipid moiety in all the GIPLs is indicated by the identification of 1 myo-inositol monophosphate residue/molecule and their susceptibility to phosphatidylinositol-specific phospholipase C. However, heterogeneity in the lipid moieties is indicated by differences in the compositional analysis and the behavior of the GIPLs on the thin layer chromatography after mild alkali hydrolysis or phospholipase A2 treatment. These results demonstrate that GIPLs 1-4 contain 1-alkyl-2-acylglycerol composed of saturated unbranched alkyl chains with carbon chain lengths of 18-26 and acyl chains of myristate, palmitate and stearate, whereas GIPL-5 and -6 contain lyso-alkylglycerol composed of mainly C24:0 and C26:0 alkyl chains. Analysis of the products of nitrous acid deamination demonstrates that these glycerolipids are present as alkylacylphosphatidylinositol (GIPLs 1-4) and 1-O-alkylglycerophosphoinositol (GIPL-5 and -6), respectively. GIPL-2 and -3 are labeled on the surface of living promastigotes with galactose oxidase/NaB[3H]4. These GIPLs also react with three monoclonal antibodies that recognize the surface of promastigotes and amastigotes of L. major and other Leishmania spp.