The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 muM) attenuated (30%; P<0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and Ad-GFPApoptin infection yielded a significant increase (P<0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.