The hydrophilic bile salt ursodeoxycholic acid (UDCA) is a potent inhibitor of apoptosis. In this paper, we further characterize the mechanism by which UDCA inhibits apoptosis induced by deoxycholic acid, okadaic acid and transforming growth factor beta1 in primary rat hepatocytes. Our data indicate that coincubation of cells with UDCA and each of the apoptosis-inducing agents was associated with an approximately 80% inhibition of nuclear fragmentation (P<0.001). Moreover, UDCA prevented mitochondrial release of cytochrome c into the cytoplasm by 70 - 75% (P<0.001), thereby, inhibiting subsequent activation of DEVD-specific caspases and cleavage of poly(ADP-ribose) polymerase. Each of the apoptosis-inducing agents decreased mitochondrial transmembrane potential and increased mitochondrial-associated Bax protein levels. Coincubation with UDCA was associated with significant inhibition of these mitochondrial membrane alterations. The results suggest that the mechanism by which UDCA inhibits apoptosis involves an interplay of events in which both depolarization and channel-forming activity of the mitochondrial membrane are inhibited.