We have previously shown that treatment of human glioma U87-MG cells expressing wild-type p53 with a DNA topoisomerase II inhibitor, etoposide resulted in ceramide-dependent apoptotic cell death. However, U87-W E6 cells lacking functional p53 due to the expression of human papilloma virus type 16 (HPV-16) E6 oncoprotein were resistant to etoposide. In order to gain insight into the roles of p53 and ceramide in gamma-radiation-induced glioma cell death, we used U87-W E6 and vector-infected U87-LXSN cells. U87-LXSN glioma cells expressing wild-type p53 were relatively resistant to gamma-radiation. U87-W E6 cells, which lost functional p53, became susceptible to radiation-induced apoptosis. Activation of caspase-3, and formation of ceramide by acid sphingomyelinase, but not by neutral sphingomyelinase, were associated with p53-independent apoptosis. Radiation-induced caspase activation and apoptotic death in U87-W E6 cells were modified by the agents which affected ceramide metabolism. SR33557, an inhibitor of acid sphingomyelinase, suppressed radiation-induced caspase activation and then apoptotic cell death. In contrast, N-oleoylethanolamine (OE) and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibit ceramidase and UDP-glucose:ceramide glucosyltransferase-1, respectively, and then augment ceramide formation, enhanced radiation-induced caspase activation. These results indicate that glioma cells with functional p53 were relatively resistant to gamma-radiation, and that ceramide may play an important role in caspase activation during gamma-radiation-induced apoptosis of glioma cells lacking functional p53.