UDP-N-acetylglucosamine:alpha(1,6)-D-mannoside beta(1,6)-N-acetylglucosaminyltransferase (GnT-V, Mgat5) functions in the biosynthesis of N-linked glycans and is transcriptionally upregulated by oncogene signaling. We report here the cloning and characterization of a human cDNA encoding a distinct enzyme with related substrate specificity, termed GnT-VB, which is predicted to have 53% similarity to the original amino acid sequence of GnT-V(A). Transient expression of GnT-VB cDNA in COS7 cells yielded significant increases of activity toward GnT-VA acceptors, including synthetic saccharides and N-linked glycopeptides, with some differences in specificity. Unlike GnT-VA, GnT-VB required divalent cation for full activity. EST databases showed expression of a 6 bp (+) splice isoform of GnT-VB; when expressed, this enzyme showed significantly reduced activity. CHO Lec4 cells, which do not express GnT-VA or B activity, lack synthesis of the N-linked beta(1,6) branch, and do not bind L-phytohemagglutinin (L-PHA), were transfected with GnT-VB or GnT-VA; both then bound significant amounts of L-PHA, demonstrating that both enzymes synthesized N-linked beta(1,6) branched glycans in vivo. Real-time polymerase chain reaction results showed that GnT-VB mRNA was highly expressed in brain and testis, with lesser levels in other tissues, while human GnT-VA showed a more general expression, but with low levels in brain and no expression in skeletal muscle.