Activator protein 2α (AP-2α) induces cytotoxicity by inducing cell cycle arrest and apoptosis. In this study we investigated the mechanism of apoptosis induction by AP-2α.We found that AP-2α induced apoptosis efficiently in cells treated with benzyloxycarbonyl-IETD-fluoromethyl ketone or FADD-silenced cells but failed to do so in benzyloxycarbonyl-LEHD-fluoromethyl ketone-treated or apoptosis protease activation factor-1 (Apaf1)-silenced cells, suggesting the central role of mitochondria in AP-2α-induced apoptosis. In good correlation, cells overexpressing AP-2α showed a reduction in mitochondrial membrane potential (Δψm), cytochrome c and Smac/DIABLO release into cytosol, and Bax translocation into mitochondria. We found that the pro-apoptotic protein Bax is important for AP-2α-induced apoptosis as adenovirus AP2 failed to induce apoptosis in HCT116 Bax -/- cells. However, we found the IAP (inhibitor of apoptosis) inhibitor Smac/DIABLO may have a limited role in AP-2α-induced apoptosis as we found the IAPmember Survivin down-regulated by AP-2α. Although the total Bax level remains unaltered, we found a time-dependent increase in the activated form of Bax in adenovirus AP2-infected cells. In addition, we show that AP-2α transcriptionally represses Bcl-2 by binding to its promoter both in vitro and in vivo and that this is essential for AP-2α-induced apoptosis as ectopic expression of Bcl-2 efficiently inhibited apoptosis induced by AP-2α. Furthermore, we show that chemotherapy-induced endogenous AP-2α down-regulates Bcl-2 and induces apoptosis in an AP-2α-dependent manner. Moreover, we demonstrate that inhibition of okadaic acid or staurosporine-sensitive pathways in AP-2αoverexpressing breast cancer cells resulted in AP-2α-dependent apoptosis induction. These results suggest that AP-2α induces apoptosis by down-regulating Bcl-2 and utilizing a bax/cytochrome c/Apaf1/caspase 9-dependent mitochondrial pathway. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.