A new beta1,4-N-acetylglucosaminyltransferase (GnT) which involves in branch formation of Asn-linked complex-type sugar chains has been purified 224,000-fold from bovine small intestine. This enzyme requires divalent cations, such as Mn2+, and catalyzes the transfer of GlcNAc from UDP-GlcNAc to biantennary oligosaccharide and produces triantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-3 arm. The purified enzyme shows a single band of Mr 58,000 and behaves as a monomer. The substrate specificity demonstrated that the beta1-2-linked GlcNAc residue on the Manalpha1-3 arm (GnT-I product) is essential for the enzyme activity. beta1-4-Galactosylaion to this essential beta1-2-linked GlcNAc residue or N-acetylglucosaminylation to the beta-linked Man residue (bisecting GlcNAc, GnT-III product) blocks the enzyme action, while beta1-6-N-acetylglucosaminylation to the Manalpha1-6 arm (GnT-V product) increases the transfer. Based on these findings, we conclude that the purified enzyme is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV (GnT-IV), that has been a missing link on biosynthesis of complex-type sugar chains.