We have previously reported the enzymatic synthesis of galactosyl-beta 1,3-N-acetylglucosamine using membrane preparations from pig trachea (Sheares, B. T., Lau, J. T. Y., and Carlson, D. M. (1982) J. Biol. Chem. 257, 599-602). The enzyme catalyzing the synthesis of this disaccharide, UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase, has been solubilized and contaminating galactosyltransferases, including the UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase and the UDP-galactose:N-acetylgalactosamine-mucin 3 beta-galactosyltransferase, were removed by affinity chromatography on alpha-lactalbumin-agarose, N-acetylglucosamine-agarose, and asialo-ovine submaxillary mucin bound to DEAE-Sephacel. UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase and a yet unidentified UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase, both not retained by the alpha-lactalbumin-agarose, were further purified by adsorption to a UDP-hexanolamine-Sepharose column and these two galactosyltransferase activities were finally resolved by gel filtration on Sephacryl S-200. The purified UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase displays an absolute requirement for a divalent cation (Mn2+ or Co2+) and is most active at pH 6.0. Unlike the UDP-galactose:N-acetylglucosamine 4 beta-galactosyltransferase, UDP-galactose:N-acetylglucosamine 3 beta-galactosyltransferase is not inhibited by high concentrations of N-acetylglucosamine. Apparent Km values are UDP-galactose, 0.23 mM, and N-acetylglucosamine, 385 mM. The apparent Km for a synthetic acceptor, N-acetylglucosaminyl-beta 1, 3-N-acetylgalactosamine-O-benzyl, was 2.4 mM. Acceptor specificity suggests that this 3 beta-galactosyltransferase is responsible for elongation of oligosaccharide chains in mucin glycoproteins and in glycolipids.