The trypanosome variant surface glycoprotein (VSG) is anchored to the outer leaflet of the parasite plasma membrane by a glycosyl phosphatidylinositol (GPI). The VSG anchor is unique among GPIs in containing exclusively dimyristoylglycerol as its lipid moiety. Myristate is incorporated into the anchor precursor by sequential deacylation and specific reacylation with myristate. Although myristate is required for the VSG anchor, trypanosomes cannot synthesize this fatty acid and must import their entire supply from the host bloodstream, where it exists in low abundance. Chemical analysis of these parasites reveals that most of their myristate is in VSG protein, with no major lipid storage form. Unexpectedly, when these cells are radiolabeled with [3H]myristate in culture, most of the label is incorporated into phospholipids, with little into VSG. This apparent contradiction is explained by the fact that trypanosomes in culture medium elongate much of the [3H]myristate into palmitate and stearate, probably because the medium (with only 5% serum) contains limiting amounts of these fatty acids. In contrast, trypanosomes radiolabeled in whole blood (with higher concentrations of palmitate and stearate) do not modify most of the [3H]myristate, and instead utilize the major portion of it for GPI synthesis. Our studies suggest that bloodstream trypanosomes have evolved highly efficient means of directing myristate into the GPI biosynthetic pathway.