The formation of tri- and tetraantennary complex-type N-linked oligosaccharides in animal glycoproteins is partly regulated by UDP-N-acetylglucosamine:beta-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase (EC 2.4.1.155) (GlcNAc-T V), which generates 2,6-branched mannose. In Chinese hamster ovary (CHO) cells we found that 2,6-branched mannosyl structures are preferentially contained on lysosome-associated membrane proteins (LAMPs) and are generally low or absent in other cellular glycoproteins (Do, K.-Y. and Cummings, R.D. (1993) J. Biol. Chem. 268, 22028-22035). To determine the mechanism by which GlcNAc-T V appears to preferentially recognize glycoproteins, we examined the activity of purified GlcNAc-T V toward a variety of glycoprotein acceptors. Because GlcNAc-T V requires as acceptors oligosaccharides lacking outer galactosyl and sialyl residues, we utilized two classes of acceptor preparations. The first class of acceptor was enzymatically desialylated (DS) and degalactosylated (DG) preparations of bovine fetuin, human transferrin, and human fibrinogen. The second class was glycoproteins in extracts of the mutant CHO cell line, Lec8 CHO, which cannot add galactose or sialic acid to N-linked oligosaccharides. GlcNAc-T V was highly active toward DSDG-fetuin, -transferrin, and -fibrinogen (Km values ranged between 30 and 74 microM), and the catalytic efficiencies (Vmax/Km) of the enzyme toward different acceptors were comparable. In the case of fetuin, each of its three sites for attachment of N-linked oligosaccharides were shown to be utilized equally well by GlcNAc-T V. Notably, the enzyme exhibited a 2-3-fold higher rate of transfer toward DSDG-transferrin when it was further denatured by reduction and S-carboxymethylation. When extracts of Lec8 CHO cells were used as acceptors, GlcNAc-T V preferentially transferred to LAMPs, and only low level transfer was observed to other cell-derived glycoproteins, thus demonstrating specificity of GlcNAc-T V toward native glycoprotein acceptors. When the cell-derived glycoproteins were denatured by reduction and S-carboxymethylation prior to use as acceptors for Glc-NAc-T V, significant transfer occurred to other glycoproteins. These results demonstrate that the mechanism of glycoprotein-specific branching by GlcNAc-T V is determined primarily by its accessibility to available bi/triantennary oligosaccharides on glycoproteins and not by its recognition of peptide determinants or conformation-specific determinants.