Abstract. African trypanosomes contain a membranebound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic Percoll R centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate.The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes.The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of 1. Abbreviations used in this paper: CRD, cross-reacting determinant; GPDH, glycerol-3-phosphate dehydrogenase; LG fraction, large granule fraction; mfVSG, membrane-attached form of the variable surface glycoprotein; SG fraction, small granule fraction; sVSG, soluble form of the variable surface glycoprotein; VSG, variable surface glycoprotein. tegrity of the surface coat is known to be essential for parasite survival in the host, the purpose of this study was to investigate VSG synthesis, processing, and replacement on the cell surface. A clearer understanding of the cell biology of the VSG may suggest alternative approaches for the possible control of trypanosomiasis.The VSGs from T. brucei are proteins that contain complex carbohydrate moeities of mannose, galactose, glucosamine, and myristilated phosphotidylinositol (17,18,39) conjugated to the protein through the alpha-carboxyl group of the carboxy-terminal amino acid via an ethanolamine residue (13,28,39). The biological importance to VSG of this carbohydrate side chain is evident since it is present on all variable antigens of T. brucei and T. congolense organisms so far studied and is probably responsible for cross-reactivity observed between soluble forms of VSGs (also known as the cross-reacting determinant [CRD]) (14). The carbohydrate side chain may be involved in anchoring the VSG to the plasma membrane (15,17,18,39), and maintaining the integrity of VSG-VSG interactions within the coat ...