We have constructed Ad CMV-Smac, a recombinant adenovirus encoding Smac/DIABLO, the recently described second mitochondrial activator of caspases. Transfection of ovarian carcinoma cells with Ad CMV-Smac at multiplicities of infection of 3-60 pfu/cell leads to increasing apoptosis in a dose-dependent manner. Western blot analysis confirms that Smac-induced apoptosis proceeds via a pathway mediated primarily by caspase-9 that can be inhibited by zLEHD-fmk and overexpression of the X-linked inhibitor of apoptosis protein (XIAP). In contrast, there is no cleavage of either caspase-8 or caspase-12. Ad CMV-Smac appears to induce apoptosis independently of cytochrome c release from mitochondria and is not inhibited by overexpression of Bcl-2. Ad CMV-Smac can combine with other proapoptotic factors, such as cisplatin, paclitaxel, and procaspase-3, to produce greater levels of apoptosis in transfected cells.