Most macromolecules at the cell surface of parasitic protozoa of the genus Leishmania, including the major surface glycoproteins and a complex lipophosphoglycan (LPG), are attached to the plasma membrane via glycosyl-phosphatidylinositol (GPI) anchors. Free glycoinositol phospholipids (GIPLs) which are not linked to protein or phosphoglycan have also been found. In this study, we show that L. mexicana promastigotes synthesize two distinct GIPL lineages, comprising at least 10 glycolipid species. These structures were characterized using a combination of gas-liquid chromatography-mass spectrometry, methylation linkage analysis, and chemical and exoglycosidase sequencing. The major lineage contains GIPLs with the glycan structures Man alpha 1-3Man alpha 1-4GlcN (iM2), Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (iM3), and Man alpha 1-2Man alpha 1-6(Man alpha 1-3)Man alpha 1-4GlcN (iM4), which are linked to alkylacyl-PI containing predominantly C16:0 and C18:0 fatty acids and C18:0 alkyl chains (referred to as the hybrid type GIPLs). A proportion of the iM3 and iM4 species (32 and 4%, respectively) are substituted with an ethanolamine-phosphate residue. The location of this residue on the core glucosamine residue was inferred from the results of methylation analyses and alpha-mannosidase digestion. The minor GIPL lineage contains GIPLs with the same glycan sequences as the glycolipid anchor of LPG (referred to as the type-2 GIPLs). The alkylacyl-PI or lyso-alkyl-PI lipid moieties of these GIPLs differ from those of the hybrid type GIPLs and from the main pool of alkylacyl-PI in containing significant levels of C24:0 and C26:0 alkyl chains. The most polar of these GIPLs, LPGp, has the properties expected of a biosynthetic precursor to the LPG, having the structure, [formula: see text] Finally, the GPI anchors of the major promastigote proteins were found to contain the glycan sequence Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN, and an alkylacyl-PI lipid moiety which was highly enriched for C24:0 or C26:0 alkyl chains. These data suggest that L. mexicana promastigotes contain three distinct pathways of GPI biosynthesis. The possibility that the distinct alkyl chain compositions of the different GPI glycolipids reflects the subcellular compartmentalization of different GPI biosynthetic pathways is discussed.