The branch specificity of bovine colostrum and calf thymus UDP-Gal:N-acetylglucosaminide beta-1----4-galactosyltransferase toward several branched oligosaccharides, which form part of the complex-type N-glycans of glycoproteins, was investigated. A novel method was used based on acetolysis of the bi[14C,3H] galactosylated oligosaccharide products formed by the enzymes in vitro and analysis of the acetolysis fragments by high-pressure liquid chromatography. It could be established that the galactosylation of different oligosaccharide branches occurred in a preferred order. No difference in branch specificity was observed between the soluble bovine colostrum galactosyltransferase and the enzyme that had been solubilized from calf thymus membranes. A preferential pathway for the biosynthesis of bisialylated biantennary glycans is proposed.