Cell lines derived from human prostate cancer are regarded as relatively resistant to both radiation-induced clonogenic death and apoptosis. Here we attempted to modulate the response of LNCaP prostate cancer cells to radiation therapy (XRT) by pretreatment with 12-O-tetradecanoylphorbol acetate (TPA), a known apoptogenic agent in LNCaP cells. Using plateau-phase cultures, we investigated the response of these cells to XRT, TPA, and a combination of XRT and TPA. LNCaP irradiation did not result in ceramide generation or apoptosis. However, pretreatment with TPA enabled XRT to generate ceramide via activation of the enzyme ceramide synthase and signal apoptosis. Apoptosis was abrogated by the competitive inhibitor of ceramide synthase, fumonisin B₁. Furthermore, when transplanted orthotopically into the prostate of nude mice, LNCaP cells produced tumors that recapitulated the responses of LNCaP cells in vitro. XRT or TPA failed to signal apoptosis in LNCaP tumors, whereas a combination of the two resulted in substantial (20–25%) apoptosis within 24 h. There was an additional benefit associated with this regimen because TPA pretreatment protected the adjacent rectum from radiation-induced apoptosis. This represents the first description of signaling-based therapy designed to overcome one form of radiation resistance expressed preferentially in LNCaP human prostate cancer cells.