UDP-GlcNAc:alpha-6-D-mannoside [GlcNAc to Man alpha 1-6] beta-1,2-N-acetylglucosaminyltransferase II (GlcNAc-T II, EC 2.4.1.143) is a Golgi enzyme catalyzing an essential step in the conversion of oligomannose to complex N-glycans. A 1.2-kb probe from a rat liver cDNA encoding GlcNAc-T II was used to screen a human genomic DNA library in lambda EMBL3. Southern analysis of restriction endonuclease digests of positive phage clones identified two hybridizing fragments (3.0 and 3.5 kb) which were subcloned into pBlueScript. The inserts of the resulting plasmids (pHG30 and pHG36) are over-lapping clones containing 5.5 kb of genomic DNA. The pHG30 insert (3.0 kb) contains a 1341-bp open reading frame encoding a 447-amino-acid protein, 250 bp of G + C-rich 5'-upstream sequence and 1.4 kb of 3'-downstream sequence. The pHG36 insert (3.5 kb) contains 2.75 kb of 5'-upstream sequence and 750 bp of the 5'-end of the open reading frame. The protein sequence showed the domain structure typical of all previously cloned glycosyltransferases, i.e. a short 9-residue putative cytoplasmic N-terminal domain, a 20-residue hydrophobic non-cleavable putative signal-anchor domain and a 418-residue C-terminal catalytic domain. Northern analysis of human tissues showed a major message at 3 kb and minor signals at 2 and 4.5 kb. There is no sequence similarity to any previously cloned glycosyltransferases including human UDP-GlcNAc:alpha-3-D-mannoside [GlcNAc to Man alpha 1-3] beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-T I) which has 445 amino acids with a 418-residue C-terminal catalytic domain. The human GlcNAc-T I and II genes (MGAT1 and MGAT2) map to chromosome bands 5q35 and 14q21, respectively, by fluorescence in situ hybridization. The entire coding regions of human GlcNAc-T I and II are each on a single exon. There is 92% identity between the amino acid sequences of the catalytic domains of human and rat GlcNAc-T II. Southern analysis of restriction enzyme digests of human genomic DNA indicates that there is only a single copy of the MGAT2 gene. The full-length coding region of GlcNAc-T II has been expressed in the baculovirus/Sf9 insect cell system, the recombinant enzyme has been purified to near homogeneity with a specific activity of about 20 mumol.min-1.mg-1 and the product synthesized by the recombinant enzyme has been identified by high-resolution 1H-NMR spectroscopy and mass spectrometry.