The death of dopaminergic and other neurons in primary cultures of the mesencephalon could be induced by treatment with ceramide, as in lymphocytes where it mediates activation by the cytokines tumor necrosis factor-alpha and interleukin-1 beta of a novel sphingomyelin-dependent signaling pathway leading to apoptosis. The morphological hallmarks of this form of cell death-bleb formation, cell body shrinkage, nuclear chromatin condensation, and fragmentation--were observed in degenerating neurons. Internucleosomal DNA degradation could also be evidenced by gel electrophoresis. The C2 and C6 analogues as well as native ceramide, administered in a dodecane suspension, had a similar effect, whereas the closely related C2-dihydroceramide, which lacks the 4-5 trans double bond in the sphingosine chain, failed to induce apoptosis. Neuronal death could be delayed by serum factors, dibutyryl cyclic AMP, and the protein synthesis inhibitor cycloheximide.