This study was undertaken to determine whether the Bcl-2 family proteins and Smac are regulators of aspirin-mediated apoptosis in a gastric mucosal cell line known as AGS cells. Cells were incubated with varying concentrations of ASA (2 to 40 mM), with or without pre-incubation of caspase inhibitors. Apoptosis was characterized by Hoechst staining and DNAhistone- associated complex formation. Anti-apoptotic Bcl-2, pro-apoptotic Bax and Bid, Smac and cytochrome c oxidase (COX IV) were analyzed by Western blots from cytosol and mitochondrial fractions. ASA down-regulated Bcl-2 protein expression, and induced Bax translocation into the mitochondria and cleavage of Bid. In contrast, expression of Smac was significantly decreased in mitochondrial fractions of ASA treated cells. Bax and Bid involvement in apoptosis regulation was dependent upon caspase activation, since caspase-8 inhibition suppressed Bax translocation and Bid processing. Caspase-9 inhibition prevented Smac release from mitochondria. Additionally, increased expression of the oxidative phosphorylation enzyme, COX IV, was observed in mitochondrial fractions exposed to ASA at concentrations higher than 5 mM. Although caspase-8 inhibition had no effect on aspirininduced apoptosis and DNA-histone complex formation, caspase-9 inhibition significantly decreased both of these events. We conclude that Bcl-2 protein family members and Smac regulate the apoptotic pathway in a caspase dependent manner. Our results indicate also that mitochondrial integration and oxidative phosphorylation play a critical role in the pathogenesis of apoptosis in human gastric epithelial cells.