Activation of the apoptosis program has been implicated in the response of cancer cells to chemotherapy. Therefore, we postulated that chemotherapy-resistant prostate cancer has developed a lesion in the apoptosis signal transduction cascade. In this study, we investigated the mechanism underlying the resistance of apoptosis-insensitive prostate cancer cells to apoptosis. We approached this by comparing the response of the androgen-sensitive LNCaP cell line and the androgen-insensitive PC3 cell line to treatment with the topoisomerase I inhibitor, camptothecin. We demonstrated that LNCaP cells are susceptible to camptothecin-induced cell death, and PC3 cells are resistant. Additional studies confirmed that the mode of cell death in the LNCaP cells was by apoptosis. We then determined that a component of the resistance to death in the apoptosis-insensitive cells involved a defect in the generation of ceramide, a key lipid mediator of apoptosis. Specifically, we demonstrated that PC3 cells are unable to elevate ceramide in response to treatment with camptothecin. In contrast, elevations in ceramide levels occur in LNCaP cells in response to the same treatment. Significantly, additional studies showed that treatment with exogenous ceramide overcomes the lesion in the PC3 cells and induces apoptosis. In attempting to gain preliminary insight into the nature of the lesion in ceramide formation in the apoptosis-resistant cells, we established that generation of ceramide in LNCaP cells is independent of the de novo pathway. These studies present novel insights into the mechanism by which prostate cancer cells may be resistant to induction of apoptosis. The significance of this study lies in the fact that an understanding of the biological and molecular events contributing to the resistance of prostate cancer to therapy is crucial to the development of more effective regimens for advanced disease.