BACKGROUND: Drug resistance is a major concern in cancer therapy. Here, we investigate the clinical potential of the second mitochondria-derived activator of caspase (Smac/DIABLO) in enhancing the apoptosis-inducing potential of commonly used anticancer drugs (paclitaxel, doxorubicin, etoposide, tamoxifen), irradiation and TRAIL in breast carcinoma. METHODS: Breast cancer cells were overexpressed with Smac/DIABLO gene (full-length or Delta55 Smac/DIABLO) or treated with Smac/DIABLO peptide to enhance the apoptosis-inducing potential of chemotherapeutic drugs and irradiation, and sensitize TRAIL-resistant cells. Cell viability and apoptosis were measured by XTT assay and DAPI staining, respectively. Protein-protein interaction was determined by immunoprecipitation followed by the Western blot analysis. RESULTS: Overexpression of Smac/DIABLO gene (full-length or Delta55 Smac/DIABLO) or treatment with Smac/DIABLO peptide enhances apoptosis induced by paclitaxel, doxorubicin, etoposide, tamoxifen, and irradiation in breast cancer cells. Overexpression of Smac/DIABLO resulted in an increased interaction of Smac/DIABLO with IAPs, which correlated with an increase in caspase-3 activity and apoptosis. Furthermore, Smac/DIABLO sensitized TRAIL-resistant breast cancer cell lines to undergo apoptosis through caspase-3 activation. These data suggest that apoptotic events down-stream of mitochondria were intact in TRAIL-resistant cells since ectopic expression of Smac/DIABLO or pretreatment of cells with Smac/DIABLO peptide completely restored TRAIL sensitivity. CONCLUSION: The ability of Smac/DIABLO agonists to enhance the apoptosis-inducing potential of chemotherapeutic drugs and irradiation, and sensitize TRAIL-resistant tumor cells suggests that Smac/DIABLO may induce fundamental alterations in cell signaling pathways. Thus, Smac/DIABLO agonists can be used as promising new candidates for cancer treatment by potentiating cytotoxic therapies.