The preceding paper (Williams, D., and Schachter, H. (1980) J. Biol. Chem. 255, 0000-0000) described a novel N-acetylglucosaminyltransferase in canine submaxillary gland microsomes which catalyzed the incorporation of GlcNAc into mucin acceptors. We show in the present report that the enzyme catalyzes th following reaction: UDP-GlcNAc + Gal beta 1-3GalNAc-X leads to Gal beta 1-3(GlcNAc beta 1-6) GalNAc-X + UDP, where X can be porcine submaxillary mucin polypeptide (Km = 5.2 mM), antifreeze glycoprotein polypeptide (Km = 23 mM), alpha-O-p-nitrophenyl (Km = 0.52 mM), beta-O-p-nitrophenyl (Km = 0.92 mM), alpha-O-o-nitrophenyl (Km = 0.86 mM), alpha-O-benzyl (Km = 0.77 mM), alpha-O-methyl (Km = 4.2 mM), or -H (Km = 1.2 mM). Ineffective acceptors (< 5% of the activity with Gal beta 1-3GalNAc-alpha-p-nitrophenyl) are asialo-ovine submaxillary mucin, asialo-alpha 1 acid glycoprotein, Gal beta 1-3GlcNAc-beta-p-nitrophenyl, Gal beta 1-3GlcNAc-alpha-methyl, Gal beta1-3GlcNAc, Gal beta 1-3-N-acetylgalactosaminitol, and D-fucose beta 1-3GalNAc-alpha-benzyl, indicating that both the Gal and GalNAc residues are essential for acceptor activity. The beta 1 leads to 6-linkage of GlcNAc to GalNAc in the acceptor Gal beta 1-3GalNAc-alpha-O-benzyl was established by methylation analysis and high resolution 1H nuclear magnetic resonance spectroscopy of a large scale preparation of product. Preliminary evidence has been obtained indicating that sialic acid linked alpha 2-6 to GalNAc or L-fucose linked alpha 1-2 to Gal inhibit the action of the beta 6-N-acetylglucosaminyltransferase on Gal beta 1-3GalNAc-porcine submaxillary mucin polypeptide.