Phosphorylation of the D-mannose residues of lysosomal enzymes is essential for the uptake and intracellular transport of these enzymes to lysosomes. The GlcNAc-P-transferase which is involved in the phosphorylation reaction seems to recognize a signal, probably a protein conformation, common to many lysosomal enzymes. To evaluate the role of the carbohydrate portion of the enzyme in these phosphorylation reactions, the acceptor specificity of GlcNAc-P-transferase from rat-liver microsomes and fibroblasts was examined with the aid of synthetic D-mannosyl disaccharides and derivatives that are closely related to the high-mannose type of oligosaccharides. Four methyl D-mannobiosides were synthesized, and their structures were established by 13C-n.m.r. spectroscopy. Of all the D-mannosyl saccharides tested, alpha-D-Man-(1----2)-alpha-D-Man-(1----OMe) was found to be the best acceptor, thereby suggesting that oligosaccharide structure may also have a role to play in recognition by this enzyme.