Gangliosides in the range of 0.1-0.4 mM inhibited the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyl transferase (EC 188.8.131.52) of chicken retina. Other lipids such as phosphatidylethanolamine, sphingomyelin, sulfatides, and phosphatidic acid in concentrations similar to those of gangliosides did not affect the enzyme activity significantly. GM3 has an inhibition capability slightly less than that of gangliosides with two or three sialyl groups in their molecules, while asialo-GM1 is clearly less inhibitory. The inhibitory effect of a constant amount of GT1 ganglioside was higher at low concentrations of membrane preparation, but the inhibition was similar at different concentrations of the substrates GM3 or UDP-N-acetylgalactosamine and at all incubation times studied. The added gangliosides were found attached to the membranes. In this attached state they may act either as substrate or inhibitor. The inhibitory effect of gangliosides was not apparent when a mixture of Triton CF 54-Tween 80 was added to the incubation medium at concentrations greater than 0.33%.