The major macromolecule on the surface of the protozoan parasite Leishmania major is a lipophosphoglycan (LPG) which contains a glycosylphosphatidylinositol glycolipid anchor. This parasite also synthesizes a complex family of abundant low-molecular-mass glycoinositolphospholipids (GIPLs) which are structurally related to the LPG anchor. In this study, L. major promastigotes were metabolically labelled with [3H]GlcN, and the kinetics of incorporation into free glycolipids and the LPG anchor followed to elucidate the pathway of GIPL biosynthesis and possible precursor-product relationships between the GIPLs and LPG. Labelled GIPLs were identified by TLC and by liquid chromatography of the released headgroups, before and after enzymic and chemical cleavage. On the basis of the measured specific radioactivities of the GIPLs, and their kinetics of radiolabelling, we suggest the pathway GlcN-PI-->Man1GlcN-PI (M1)-->Man2GlcN-PI (iM2)-->GalfMan2GlcN-PI (GIPL-1)-->Gal1GalfMan2GlcN-PI (GIPL-2)-->Gal2GalfMan2GlcN-PI (GIPL-3). All of the GIPLs were shown to contain alkylacylglycerol or lyso-alkylglycerol lipid moieties with the exception of the earliest intermediate, glucosaminylphosphatidylinositol (GlcN-PI), which contained both alkylacylglycerol and diacylglycerol. A significant proportion (approx. 50%) of GIPL-3 appeared to be selectively modified by the addition of a Glc-1-PO4 residue to one of the mannose residues (P-GIPL-3). On the basis of the specific radioactivity and kinetics of labelling of GIPL-3 and P-GIPL-3 we suggest that both of these low-abundance species are rapidly utilized as LPG precursors. The turnover of LPG and the GIPLs was also studied by [3H]Gal pulse-chase labelling and cell-surface labelling experiments. Whereas LPG was rapidly shed from the cell surface, consistent with previous studies, the GIPLs (both the total cellular and cell-surface pools) had a much slower turnover. These results suggest that the majority of the GIPLs do not act as LPG precursors and indicate that the cellular levels of these molecules is determined, at least in part, by the rate at which they are shed from the cell surface.