Glycosylphosphatidylinositol (GPI) membrane anchors are ubiquitous among the eukaryotes. In most organisms, the pathway of GPI biosynthesis involves inositol acylation and inositol deacylation as discrete steps at the beginning and end of the pathway, respectively. The bloodstream form of the protozoan parasite Trypanosoma brucei is unusual in that these reactions occur on multiple GPI intermediates and that it can express side chains of up to six galactose residues on its mature GPI anchors. An inositol deacylase gene, T brucei GPIdeAc, has been identified. A null mutant was created and shown to be capable of expressing normal mature GPI anchors on its variant surface glycoprotein. Here, we show that the null mutant synthesizes galactosylated forms of the mature GPI precursor, glycolipid A, at an accelerated rate (2.8-fold compared to wild type). These free GPIs accumulate at the cell surface as metabolic end products. Using continuous and pulse-chase labeling experiments, we show that there are two pools of glycolipid A. Only one pool is competent for transfer to nascent variant surface glycoprotein and represents 38% of glycolipid A in wild-type cells. This pool rises to 75% of glycolipid A in the GPIdeAc null mutant. We present a model for the pathway of GPI biosynthesis in T brucei that helps to explain the complex phenotype of the GPIdeAc null mutant.