The membrane form of Trypanosoma brucei variant surface glycoprotein (mfVSG) is acylated with ester-linked tetradecanoic (myristic) acid (Ferguson, M. A. J., and Cross, G. A. M. (1984) J. Biol. Chem. 259, 3011-3015). Comparative analysis of Pronase peptides from mfVSG and soluble VSG localizes the site of mfVSG acylation to a COOH-terminal oligosaccharide structure. Chemical and enzymatic treatment of the acylated Pronase mfVSG fragment revealed that the myristic acid is present as a diglyceride (sn-1,2-dimyristin) that is probably linked to the COOH-terminal oligosaccharide via a phosphodiester bond between the sn-3-glycerol hydroxyl and a sugar hydroxyl group. The endogenous membrane-associated enzyme, which quantitatively cleaves myristic acid from mfVSG to produce soluble VSG, releases diglyceride, as would be expected of a phospholipase C.