Glycolipid A, the precursor of the glycosyl-phosphatidylinositol (GPI) anchor of the trypanosome variant surface glycoprotein, is constructed in two phases. First, the glycan is assembled on phosphatidylinositol (PI), yielding a glycolipid termed A'. Second, glycolipid A' undergoes fatty acid remodelling, by deacylation and reacylation, to become the dimyristoyl species glycolipid A. In this paper, we examine the fatty acid content of glycolipid A' and its cellular progenitors. A' contains exclusively stearate at the sn-1 position and a complex mixture of fatty acids (including 18:0, 18:1, 18:2, 20:4 and 22:6) at sn-2. Presumably these fatty acids derive from stearate-containing PI species which initially enter the biosynthetic pathway. We compared the diacylglycerol species from glycolipid A' with those from phosphatidylinositol to determine whether a subset of stearate-containing PIs is utilized for GPI biosynthesis. We found that the spectrum of stearate-containing diacylglycerols in PI is similar to that in A', although the proportions of each compound differ. Total PI in general was highly enriched in stearate-containing species. Differences in composition between glycosylated PI and total cellular PI may be due to the substrate specificity of the sugar transferase which initiates the GPI biosynthetic pathway. Alternatively, the species of PI present at the endoplasmic reticulum site of GPI biosynthesis may differ from those in total PI.