Glycosylphosphatidylinositol precursor-analogues were synthesized in which the natural diacylglycerol lipid was replaced with either of two steroidal moieties. The ability of the steroidal glycosylphosphatidylinositol precursor-analogues to prime the glycosylphosphatidylinositol biosynthetic pathway was assessed in a trypanosomal cell-free system. The N-acetyl-D-glucosaminylphosphatidylinositol de-N-acetylase was only able to act upon the N-acetylglucosamine form of one of the two analogues. However, the glucosamine form of both analogues could be mannosylated, but neither were inositol-acylated nor modified with ethanolamine phosphate. The use of alternative groups, such as sterols, in place of the natural diacylglycerol lipid may enable the production of more drug-like, substrate-based inhibitors.