Purpose: TRAIL/Apo-2L is cytotoxic against numerous prostate tumor cell lines; however, some lines are more resistant than others. Identification of an agent that increases prostate tumor cell sensitivity to TRAIL/Apo-2L would prove valuable for TRAIL/Apo-2L-mediated tumor therapy. Thus, we examined the effect of combining five clinically approved chemotherapeutic agents with TRAIL/Apo-2L for treating prostate tumor cells. Methods: Four human prostate tumor cell lines were initially tested for TRAIL/Apo-2L sensitivity. Subsequent studies examined whether the TRAIL/Apo-2L-induced killing of DU-145 cells was augmented in the presence of the chemotherapeutic molecules, as measured by annexin V-FITC/propidium iodide staining. Furthermore, caspase 8 activation and BID cleavage were examined by immunoblotting. RT-PCR and flow cytometry were performed to monitor TRAIL-R1 and TRAIL-R2 levels after chemotherapeutic treatment. Results: DU-145 cells were the least responsive of the prostate tumor cell lines tested to TRAIL/Apo-2L-induced death, Surprisingly, only topotecan, a topoisomerase I inhibitor, when used in combination with rTRAIL/Apo-2L led to significant apoptosis of DU-145 cells, as measured by caspase 8 activation, BID cleavage, and annexin V-FITC/PI staining. Topotecan alone had little to no toxicity on the DU-145 cells. Furthermore, the increase in TRAIL/Apo-2L sensitivity following topotecan treatment correlated with increased expression of TRAIL-R1 and TRAIL-R2 and decreased intracellular levels of the antiapoptotic protein survivin. Conclusions: Our results define a promising direction for alternative therapies against androgen-independent prostate cancers. The sensitivity of DU-145 cells to TRAIL/Apo-2L was dramatically increased when combined with topotecan, suggesting that low-dose topotecan treatment to upregulate TRAIL-R1 and TRAIL-R2 and downregulate survivin, followed by TRAIL/Apo-2L administration, may be a viable therapy for treating cancer of the prostate.