Ceramide has been implicated as an important component of radiation-induced apoptosis of human prostate cancer cells. We examined the role of the sphingolipid metabolites—ceramide, sphingosine, and sphingosine–1-phosphate—in susceptibility to radiation-induced apoptosis in prostate cancer cell lines with different sensitivities toγ-irradiation. Exposure of radiation-sensitive TSU-Pr1 cells to 8-Gy irradiation led to a sustained increase in ceramide, beginning after 12 h of treatment and increasing to 2.5- to 3-fold within 48 h. Moreover, irradiation of TSU-Pr1 cells also produced a marked and rapid 50% decrease in the activity of sphingosine kinase, the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate. In contrast, the radiation-insensitive cell line, LNCaP, had sustained sphingosine kinase activity and did not produce elevated ceramide levels on 8-Gy irradiation. Although LNCaP cells are highly resistant to γ-irradiation-induced apoptosis, they are sensitive to the death-inducing effects of tumor necrosis factor α,which also increases ceramide levels in these cells (K. Kimura et al., Cancer Res., 59: 1606–1614,1999). Moreover, we found that although irradiation alone did not increase sphingosine levels in LNCaP cells, tumor necrosis factor αplus irradiation induced significantly higher sphingosine levels and markedly reduced intracellular levels of sphingosine-1-phosphate. The elevation of sphingosine levels either by exogenous sphingosine or by treatment with the sphingosine kinase inhibitor N,N-dimethylsphingosine induced apoptosis and also sensitized LNCaP cells to γ-irradiation-induced apoptosis. Our data suggest that the relative levels of sphingolipid metabolites may play a role in determining the radiosensitivity of prostate cancer cells, and that the enhancement of ceramide and sphingosine generation could be of therapeutic value.