Ganglioside distribution in cells undergoes deep modifications during physiological and pathological events, possibly depending on the activity of glycosyltransferases involved in their biosynthesis. To understand how the ganglioside pattern can be altered by the selective expression of specific glycosyltransferases, C6 rat glioma cell line was stably transfected with two human glycosyltransferase cDNAs: β-1,4N-acetylgalactosaminyltransferase (GaINAcT) and a-2,8-sialyltransferase (ST-II). GaINAcT and ST-II are key enzymes in ganglioside biosynthesis; whereas ST-II synthesizes GD 3 , precursor of the b pathway, GaINAcT produces GM 2 , GD 2 and asialo-GM 2 and it is, therefore, involved in a, b and asialo pathways. C6 cells were subjected to three independent transfections: one with a construct containing GaINAcT cDNA, one with a construct containing the ST-II cDNA, and one with both constructs simultaneously. Whereas control cells present mainly N-acetyl- and N-glycolyl-GM 3 , selected transfected clones show more complex ganglioside profiles: GaINAcT-expressing cells are enriched in the a series gangliosides, ST-II-expressing cells synthesize the b series species, cells expressing contemporarily the two glycosyltransferases produce gangliosides of both series. Furthermore, among the selected clones, expression of GaINAcT and ST-II correlates with changes in the ST-I and ST-IV activities, indicating that the switching on of the biosynthetic enzymes we investigated influences the activity of endogenous glycosyltransferases, possibly through the modification of the amount of their substrates or products.