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 alpha-mannosides and octyl alpha-mannosides all accepted single mannose residues in alpha-linkage, as judged by thin layer chromatography of the products before and after jack bean alpha-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 alpha-mannose residues were shown to be both alpha 1-2 and alpha 1-6 linked by Aspergillus phoenicis alpha-mannosidase and acetolysis treatments, respectively. These data suggest that the compounds can act as acceptor substrates for the Dol-P-Man dependent alpha 1-2 and alpha 1-6 mannosyltransferases of the GPI biosynthetic pathway and/or the dolichol-cycle of protein N-glycosylation. One of the compounds, Man alpha 1-6 Man alpha 1-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-Pl alpha 1-2 mannosyltransferase.