A membrane-associated galactosyltransferase from Trypanosoma brucei was purified 34000-fold by affinity chromatography on UDP-hexanolamine–Sepharose™. Using SDS/PAGE under reducing conditions, the isolated enzyme ran as a relatively broad band with apparent molecular masses of 53 kDa and 52 kDa, indicative of glycosylation and the existence of two isoforms. N -Glycosylation of the enzyme was subsequently confirmed using Western blotting and either specific binding of concanavalin A or peptide- N 4 -( N -acetylglucosaminyl)asparagine amidase digestion. The de- N -glycosylated enzyme ran with apparent molecular masses of 51 kDa and 50 kDa, indicative of a single N -glycosylation site. The galactosyltransferase exhibited a pH optimum at 7.2 and had a pronounced requirement for Mn 2+ ions ( K M = 2.5 mM) for its action. The transferase activity was independent of the concentration of Triton X-100. The enzyme was capable of transferring galactose from UDP-galactose to a variety of galactose-based acceptors in α-glycosidic linkages. The apparent K M values for UDP-galactose and for the preferred acceptor substrate N -acetyl-lactosamine are 46 µ M and 4.5 mM respectively. From these results we would like to suggest that the galactosyltransferase functions in the processing of terminal N -acetyl-lactosamine structures of trypanosomal glycoproteins.