We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137-145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from either l-[(3)H]serine or [(3)H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.