Ceramides are well-known second messengers that induce apoptosis in various kinds of cancer cells, and their effects are closely related to radiation sensitivity. Phytoceramides, the yeast counterparts of the mammalian ceramides, are also reported to induce apoptosis. We investigated the effect of a novel ceramide derivative, N-acetylphytosphingosine (NAPS), on the radiosensitivity of NCI-H460 human lung carcinoma cells and its differential cytotoxicity in tumor and normal cells. The combination of NAPS with radiation significantly increased clonogenic cell death and caspase-dependent apoptosis. The combined treatment greatly increased Bax expression and Bid cleavage, but not Bcl-2 expression. However, there was no effect on radiosensitivity and apoptosis in BEAS2B cells, which derive from normal human bronchial epithelium. Cell proliferation and DNA synthesis were significantly inhibited by NAPS in both NCI-H460 and BEAS2B cells, but only the BEAS2B cells recovered by 48h after removal of the NAPS. Furthermore, the NCI-H460 cells underwent more DNA fragmentation than the BEAS2B cells in response to NAPS. Our results indicate that NAPS may be a potential radiosensitizing agent with differential effects on tumor vs. normal cells.