Sphingolipids are abundant constituents of neuronal membranes and have been implicated in intracellular signaling. We show that two analogs of glycosphingolipid biosynthetic intermediates, fumonisin B1 (which inhibits dihydroceramide synthesis) and DL-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) (which inhibits glucosylceramide synthesis) decrease glycosphingolipid synthesis in rat sympathetic neurons. Although both fumonisin and PPMP inhibit glycosphingolipid synthesis, these inhibitors have differential effects on ceramide metabolism in axons. threo-PPMP, but not erythro-PPMP or fumonisin, induces an accumulation of [3H]palmitate-labeled ceramide and impairs axonal growth. Moreover, exogenously added, cell-permeable C6-ceramide, but not C6-dihydroceramide, mimicks the effect of PPMP. Our studies suggest that the lipid second messenger ceramide acts in distal axons, but not cell bodies, as a negative regulator of neurite growth.