To enhance the killing effects of ionizing radiation, we amplified the endogenous ceramide signal in Jurkat cell cultures using 3 different inhibitors of sphingolipid metabolism: DL-PDMP, D-MAPP and imipramine. Of the various possible drug combinations, only DL-PDMP (20 μM) + imipramine (20 μM) and DL-PDMP (20 μM) + imipramine (20 μM) + D-MAPP (5 μM) induced a major increase in ceramide levels, reaching 240% and 340% of control values, respectively, after incubation for 48 hr. With these models, we demonstrate that endogenously formed ceramide triggers time- and concentration-dependent apoptosis through induction of mitochondrial injury and activation of the caspase pathway. Cellular dysfunction includes alterations to the cellular redox potential, as assessed by the generation of ROS and total glutathione depletion, and a drop in ΔΨm. A parallel elevation of mitochondrial ceramide levels was also observed. The combination of DL-PDMP + imipramine ± D-MAPP with 10 Gy irradiation produced cumulative effects leading to apoptosis via mitochondrial collapse and activation of the caspase cascade. The association efficiency was confirmed in normal and acid sphingomyelinase-deficient lymphoid cell lines. Taken together, these results suggest that increasing endogenous ceramide levels may potentially be very valuable when combined with ionizing radiation in tumor therapy. © 2002 Wiley-Liss, Inc.