A series of hydrophobic mannosides were synthesized and tested for their ability to act as acceptor substrates for mannosyltransferases in a Trypanosoma brucei cell-free system. The thiooctyl a-mannosides and octyl a-mannosides all accepted single mannose residues in a-linkage, as judged by thin layer chromatography of the products before and after jack bean a-mannosidase digestion. The mannosylation reactions were inhibited by amphomycin, suggesting that the immediate donor was dolichol-phosphate-mannose (Dol-P-Man) in all cases. The transferred a-mannose residues were shown to be both a1-2 and a1-6 linked by Aspergillus phoenicis a-mannosidase and acetolysis treatments, respectively. These data suggest that the compounds can act as acceptor substrates for the Dol-P-Man dependent a1-2 and a1-6 mannosyltransferases of the GPI biosynthetic pathway and/or the dolichol-cycle of protein N-glycosylation. One of the compounds, Mana1-6Mana1-O-(CH2)7CH3, inhibited endogenous GPI biosynthesis in the cell-free system, suggesting that it could be a substrate for the trypanosome Dol-P-Man:Man2GlcN-Pla1-2 mannosyltransferase.