Abstract Membrane-bound α- and β- N-acetylgalactosaminyltransferases (EC 188.8.131.52) which catalyze formation of non-reducing terminal linkages of Forssman hapten and globoside, respectively, could be differentiated with respect to the different effects of UDP on the two enzyme activities. UDP markedly inhibited the α-transferase activity, in contrast to its stimulatory action on the β-transferase. These effects of UDP were similar to those of UDPglucose, which was demonstrated to be a competitive inhibitor ( K i, 3.3·10 −5 M for UDP- N-acetylgalactosamine) for the α-transferase reaction. Other uridine derivatives tested suppressed both the transferase activities, being more inhibitory for the α-transferase than for the β-transferase. Under the synthetic conditions of these aminoglycolipids, UDP- N-acetylgalactosamine as a donor was simultaneously degraded into N-acetylgalactosamine-1-phosphate and finally into N-acetylgalactosamine by UDP- N-acetylgalactosamine pyrophosphatase, which is part of the membrane system. UDP-glucose was confirmed as being able to prevent the enzymatic hydrolysis of UDP- N-acetylgalactosamine. UDPglucose, therefore, acts to suppress both the α- N-acetylgalactosaminyltransferase (but not the β-transferase) and the pyrophosphatase activities. The inhibitory effect of UDPglucose on the α-transferase activity was most probably due to its direct action on the transferase rather than its function in protecting UDP- N-acetylgalactosamine donor from pyrophosphatase action.