Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various tumor cells. The anthracycline doxorubicin (DOX) can sensitize several types of cancer cells to TRAIL-mediated apoptosis. Here we report that DOX enhances TRAIL-induced apoptosis and cytotoxicity against prostate cancer cells. Cytotoxicity was determined by a MTT assay. Synergistic effect was assessed by isobolographic analysis. Caspase activity was determined by a quantitative colorimetric assay. The combination treatment with DOX and TRAIL resulted in a synergistic cytotoxic effect on LNCaP, LNCaP-Bcl-2, PC-3, and PC93 human prostate cancer cell lines, but not on normal human prostatic stromal cells. Synergistic cytotoxicity was also obtained even when the exposure time was shortened from 24 to 8 or 2 h. A similar effect was achieved with TRAIL in combination with epirubicin, pirarubicin, or amrubicin. The synergy obtained in cytotoxicity with TRAIL and DOX was also achieved in apoptosis. DOX treatment significantly activated caspase-8, -6, and -3 in LNCaP cells. Furthermore, the synergistic cytotoxicity of TRAIL and DOX was completely inhibited by Z-VAD-FMK, and partly inhibited by Ac-IETD-CHO, Ac-DQTD-CHO, or Ac-DMQD-CHO. These findings indicate that DOX enhances TRAIL-induced apoptosis and cytotoxicity in prostate cancer by activation of caspase cascades, and suggest that TRAIL in combination with DOX have a therapeutic potential in the treatment of prostate cancer.