The precursor of the glycosylphosphatidylinositol membrane anchor of the variant surface glycoprotein of Trypanosoma brucei is known as glycolipid A and it has the structure: EtN-PO4-6Mana1-2Mana1-6Mana1-4GlcNa1-6myo-inositoll-PO4-(sn-1,2-dimyristoylglycerol). This precursor exists in equilibrium with its inositol-acylated form known as glycolipid C that contains a fatty acid attached to the inositol ring. In this study, we describe the purification to homogeneity of glycolipid C, its precise quantification and the analysis of the molecular species of glycolipid C by electrospray ionisation mass spectrometry. The results show that glycolipid C is present at 160 000 copies per cell, that glycolipid C is acylated on the 2-position of the myo-inositol ring and that glycolipid C is heterogeneous with respect to the acyl chain attached to the inositol ring. The implications of these results with respect to the nature of the trypanosome inositol acyltransferase are discussed.