The accumulation of hydrophobic bile acids plays a role in the induction of apoptosis and necrosis of hepatocytes during cholestasis. The aim of this study was to determine in freshly isolated rat hepatocytes the roles of oxidant stress and the mitochondrial permeability transition (MPT) in bile acid-induced apoptosis. Hepatocytes isolated from adult male Sprague-Dawley rats were incubated for 4 hours in buffer containing the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC, 0-500 micromol/L) or the hydrophilic bile acid, glycocholic acid (GCA), and either the antioxidants, alpha tocopherol, ebselen, or idebenone (a coenzyme Q analogue); or the MPT blockers, cyclosporin A, or bongkrekic acid, or a caspase-8 inhibitor. Apoptosis was assessed hourly by nuclear morphologic changes of fixed cells by DAPI fluorescence microscopy and reactive oxygen species (ROS) generation by dichlorofluorescein fluorescence of hepatocytes. The percent of cells undergoing apoptosis increased in a time- and concentration-dependent manner in cells exposed to GCDC, and to a much lesser extent to GCA. ROS generation preceded the onset of apoptosis. MPT blockers, caspase-8 inhibition, and antioxidants prevented apoptosis and reduced ROS generation by hepatocytes. Flow cytometry analysis showed that MPT occurred within 1 hour of exposure of cells to 100 micromol/L GCDC, prior to onset of significant apoptosis. In conclusion, ROS generation, MPT induction, and cytochrome c release are critical steps in the induction of apoptosis by bile acids. Antioxidants may reduce liver injury caused by low levels of bile acids by preventing the generation of oxidant stress and subsequent stimulation of the MPT and release of cytochrome c from mitochondria.