A mannosyltransferase that catalyzes transfer from GDP-mannose to tetrasaccharide-pyrophosphoryl-lipid with the formation of the alpha-1,3-mannosyl-mannose linkage in Man alpha 1-3(Man alpha 1-6)Man beta-GlcNAc beta-GlcNAc-P-P-lipid has ben purified 660-fold from rabbit liver microsomes. The enzyme was completely separated from mannosyltransferases that synthesize alpha-1,2-mannosyl-mannose linkages, but the purified preparation still contained some activity which synthesized alpha-1,6-mannosyl linkages. The enzyme has a requirement for divalent cations and a pH optimum between 6.8 and 7.3, and the purified enzyme was very sensitive to detergent concentration with optimal activity at 0.0225% Nonidet P-40. The extent of purification of the enzyme and its resistance to inhibition by amphomycin strongly suggest that the enzyme catalyzes direct transfer from nucleotide-sugar to the oligosaccharide-lipid acceptor.