Sphingomyelin hydrolysis and ceramide generation have been implicated in a signal transduction pathway that mediates the effects of tumor necrosis factor-α (TNF-α) and other agents on cell growth and differentiation. In many leukemic cells, TNF-α causes DNA fragmentation, which leads to programmed cell death (apoptosis). C₂-ceramide (0.6 to 5 μM), a synthetic cell-permeable ceramide analog, induced internucleosomal DNA fragmentation, which was inhibited by zinc ion. Other amphiphilic lipids failed to induce apoptosis. The closely related C₂-dihydroceramide was also ineffective, which suggests a critical role for the sphingolipid double bond. The effects of C₂-ceramide on DNA fragmentation were prevented by the protein kinase C activator phorbol 12-myristate 13-acetate, which suggests the existence of two opposing intracellular pathways in the regulation of apoptosis.