Hen oviduct membranes have been shown to catalyze the transfer of GlcNAc from UDP-GlcNAc to GlcNAc-beta 1-2Man alpha 1-6(GlcNAc beta 1-2 Man alpha 1-3) Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X (GnGn) to form the triantennary structure GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. The enzyme has been named UDP-GlcNAc:GnGn (GlcNAc to Man alpha 1-3) beta 4-N-acetylglucosaminyltransferase IV (GlcNAc-transferase IV) to distinguish it from three other hen oviduct GlcNAc-transferases designated I, II, and III. Since GlcNAc-transferases III and IV both act on the same substrate, concanavalin A/Sepharose was used to separate the products of the two enzymes. At pH 7.0 and at a Triton X-100 concentration of 0.125% (v/v), GlcNAc-transferase IV activity in hen oviduct membranes is 7 nmol/mg of protein/h. The product was characterized by high resolution proton NMR spectroscopy at 360 MHz and by methylation analysis. In addition to triantennary oligosaccharide, hen oviduct membranes produced about 20% of bisected triantennary material, GlcNAc beta 1-2Man alpha 1-6[GlcNAc beta 1-2(GlcNAc beta 1-4)Man alpha 1-3] [GlcNAc beta 1-4]Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-X. Maximal GlcNAc-transferase IV activity requires the presence of both terminal beta 1-2-linked GlcNAc residues in the substrate. Removal of the GlcNAc residue on the Man alpha 1-6 arm or of both GlcNAc residues reduces activity by at least 80%. A Gal beta 1-4GlcNAc disaccharide on the Man alpha 1-6 arm reduces activity by 68% while the presence of this disaccharide on the Man alpha 1-3 arm reduces activity to negligible levels. A similar substrate specificity was found for GlcNAc-transferase III, the enzyme which adds a bisecting GlcNAc in beta 1-4 linkage to the beta-linked Man residue. Since a bisecting GlcNAc was found to prevent GlcNAc-transferase IV action, the bisected triantennary material found in the incubation must have been formed by the sequential action of GlcNAc-transferase IV followed by GlcNAc-transferase III. Activities similar to GlcNAc-transferase IV were also detected in rat liver Golgi-rich membranes (0.4 nmol/mg/h) and pig thyroid microsomes (0.1 nmol/mg/h).