The core 3 structure of the O-glycan, GlcNAcbeta1-3GalNAcalpha1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide beta1,3-N- acetylglucosaminyltransferase (beta3Gn-T; core 3 synthase). The core 3 structure is restricted in its occurrence to mucins from specific tissues such as the stomach, small intestine, and colon. A partial sequence encoding a novel member of the human beta3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it beta3Gn-T6. The putative amino acid sequence of beta3Gn-T6 retains the beta3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of beta3Gn-T6 expressed in insect cells showed beta3Gn-T activity toward GalNAcalpha-p-nitrophenyl and GalNAcalpha1-serine/threonine. The beta1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. beta3Gn-T6 effectively transferred a GlcNAc to the GalNAc residue on MUC1 mucin, resulting in the synthesis of a core 3 structure. Real time PCR analysis revealed that the beta3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that beta3Gn-T6 is the most logical candidate for the core 3 synthase, which plays an important role in the synthesis of mucin-type O-glycans in digestive organs.