Previous studies demonstrated that inhibition of sphingolipid synthesis by the mycotoxin fumonisin B1 (FB1) disrupts axonal growth in cultured hippocampal neurons (Harel, R., and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481) by affecting the formation or stabilization of axonal branches (Schwarz, A., Rapaport, E., Hirschberg, K., and Futerman, A.H. (1995) J. Biol. Chem. 270, 10990-10998). We now demonstrate that long term incubation with FB1 affects fibroblast morphology and proliferation. Incubation of Swiss 3T3 cells with FB1 resulted in a decrease in synthesis of ganglioside GM3, the major glycosphingolipid in 3T3 fibroblasts and of sphingomyelin. The projected cell area of FB1-treated cells was approximately 45% less than control cells. FB1 had no affect on the organization of microtubules or intermediate filaments, but fewer actin-rich stress fibers were observed, and there was a loss of actin-rich lamellipodia at the leading edge. Three other processes involving the actin cytoskeleton, cytokinesis, microvilli formation, and the formation of long processes induced by protein kinase inhibitors, were all disrupted by FB1. All the effects of FB1 on cell morphology could be reversed by addition of ganglioside GM3 even in the presence of FB1, whereas the bioactive intermediates, sphinganine, sphingosine, and ceramide, were without effect. Finally, FB1 blocked cell proliferation and DNA synthesis in a reversible manner, although ganglioside GM3 could not reverse the effects of FB1 on cell proliferation. Together, these data suggest that ongoing sphingolipid synthesis is required for the assembly of both new membrane and of the underlying cytoskeleton.