[en] The capacity of UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAc) as an in vitro acceptor substrate for beta-1,4-galactosyltransferase (beta4GalT1, EC 126.96.36.199) from human and bovine milk and for recombinant human beta4GalT1, expressed in Saccharomyces cerevisiae, was evaluated. It turned out that each of the enzymes is capable to transfer Gal from UDP-alpha-D-galactose (UDP-Gal) to UDP-GlcNAc, affording Gal(beta1-4)GlcNAc(alpha1-UDP (UDP-LacNAc). Using beta4GalT1 from human milk, a preparative enzymatic synthesis of UDP-LacNAc was carried out, and the product was characterized by fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy. Studies with all three beta4GalTs in the presence of alpha-lactalbumin showed that the UDP-LacNAc synthesis is inhibited and that UDP-alpha-D-glucose is not an acceptor substrate. This is the first reported synthesis of a nucleotide-activated disaccharide, employing a Leloir glycosyltransferase with a nucleotide-activated monosaccharide as acceptor substrate. Interestingly, in these studies beta4GalT1 accepts an alpha-glycosidated GlcNAc derivative. The results imply that beta4GalT1 may be responsible for the biosynthesis of UDP-LacNAc, previously isolated from human milk.