Human and mouse cDNAs encoding a new β-1,3- N -acetylglucosaminyltransferase (β3GnT) have been isolated from fetal and newborn brain libraries. The human and mouse cDNAs included ORFs coding for predicted type II transmembrane polypeptides of 329 and 325 aa, respectively. The human and mouse β3GnT homologues shared 90% similarity. The β3GnT gene was widely expressed in human and mouse tissues, although differences in the transcript levels were visible, thus indicating possible tissue-specific regulation mechanisms. The β3GnT enzyme showed a marked preference for Gal(β1–4)Glc(NAc)-based acceptors, whereas no activity was detected on type 1 Gal(β1–3)GlcNAc and O-glycan core 1 Gal(β1–3)GalNAc acceptors. The new β3GnT enzyme was capable of both initiating and elongating poly- N -acetyllactosamine chains, which demonstrated its identity with the poly- N -acetyllactosamine synthase enzyme (E.C. 2.4.1.149), showed no similarity with the i antigen β3GnT enzyme described recently, and, strikingly, included several amino acid motifs in its protein that have been recently identified in β-1,3-galactosyltransferase enzymes. The comparison between the new UDP–GlcNAc:βGal β3GnT and the three UDP–Gal:βGlcNAc β-1,3-galactosyltransferases-I, -II, and -III reveals glycosyltransferases that share conserved sequence motifs though exhibiting inverted donor and acceptor specificities. This suggests that the conserved amino acid motifs likely represent residues required for the catalysis of the glycosidic (β1–3) linkage.