Calf thymus was found to contain a high activity of a galactosyltransferase, which transfers galactose from UDP-galactose to asialo-alpha 1-acid glycoprotein and N-acetyllactosamine. By means of competition and acceptor-specificity studies the enzyme could be distinguished from an N-acetylglucosaminide beta-1,4-galactosyltransferase and an N-acetylgalactosamine-protein beta-1,3-galactosyltransferase, which in addition occur in calf thymus, as well as from the blood-group-B-associated alpha-galactosyltransferase. Identification of the products revealed that the enzyme accomplishes an alpha 1 leads to 3 linkage resulting in a terminal Gal(alpha 1 leads to 3)Gal(beta 1 leads to 4)GlcNAc sequence. The enzyme is membrane-bound and is activated by Triton X-100. It shows optimal activity over a broad pH range (5.5-7.0) and has a pronounced requirement for Mn2+ ions (Km = 6.1 mM) for its action. It is suggested that the alpha-1,3-galactosyltransferase functions in the biosynthesis of calf thymocyte cell-surface glycoconjugates including glycoproteins.