Ceramide is a key lipid molecule necessary to regulate some cellular processes, including apoptosis and cell differentiation. In this context, its production has been shown to occur via sphingomyelin hydrolysis or sphingosine acylation. Here, we show that in human fibroblasts, plasma membrane ceramide is also produced from ganglioside GM3 by detachment of sugar units. Membrane-bound glycosylhydrolases have a role in this process. In fact, the production of ceramide from GM3 has been observed even under experimental conditions able to block endocytosis or lysosomal activity, and the overexpression of the plasma membrane ganglioside sialidase Neu3 corresponded to a higher production of ceramide in the plasma membrane. The increased activity of Neu3 was paralleled by an increase of GM3 synthase mRNA and GM3 synthase activity. Neu3-overexpressing fibroblasts were characterized by a reduced proliferation rate and higher basal number of apoptotic cells in comparison with wild-type cells. A similar behavior was observed when normal fibroblasts were treated with exogenous C₂-ceramide.--Valaperta, R., Chigorno, V., Basso, L., Prinetti, A., Bresciani, R., Preti, A., Miyagi, T., and Sonnino, S. Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts.