We found a novel human glycosyltransferase gene carrying a hypothetical beta1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5'-rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank(TM) accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc beta-benzyl. The product was deduced to be GalNAcbeta1-4GlcNAcbeta-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme beta1,4-N-acetylgalactosaminyltransferase-III (beta4GalNAc-T3). beta4GalNAc-T3 effectively synthesized N,N'-diacetylgalactosediamine, GalNAcbeta1-4GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N, N'-diacetylgalactosediamine structures in their N-glycans, we examined the ability of beta4GalNAc-T3 to synthesize N, N'-diacetylgalactosediamine structures in N-glycans on a model protein. When fetal calf fetuin treated with neuraminidase and beta1,4-galactosidase was utilized as an acceptor protein, beta4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that beta4GalNAc-T3 could transfer GalNAc residues, producing N,N'-diacetylgalactosediamine structures at least in N-glycans and probably in both N- and O-glycans.