A series of synthetic mannosides was screened in a cell-free system for their ability to act as acceptor substrates for mycobacterial mannosyltransferases. Evaluation of these compounds demonstrated the incorporation of [14C]Man from GDP-[14C]Man into a radiolabeled organic-soluble fraction and analysis by thin layer chromatography and autoradiography revealed the formation of two radiolabeled products. Each synthetic acceptor was capable of accepting one or two mannose residues, resulting in a major and a minor mannosylated product. Both products from each acceptor were isolated and their mass was confirmed by fast-atom bombardment-mass spectrometry (FABMS). Characterization of each mannosylated product by exo-glycosidase digestion. acetolysis and linkage analysis by gas chromatography mass spectrometry of partially per-O-methylated alditols, revealed only alpha1-6-linked products. In addition. the antibiotic amphomycin selectively inhibited the formation of mannosylated products suggesting polyprenolmonophosphate-mannose (C15 50-P-Man) was the immediate mannose donor in all mannosylation reactions observed. The ability of synthetic disaccharides to act as acceptor substrates in this system, is most likely due to the action of a mycobacterial polyprenol-P-Man:mannan alpha1-6 mannosyltransferase involved in the biosynthesis of linear alpha1-6-linked lipomannan.