The therapeutic efficacy of ursodeoxycholic acid (UDCA) has been widely demonstrated in various liver diseases, suggesting that UDCA might protect hepatocytes against common mechanisms of liver damage. A candidate for such protection is oxidative injury induced by reactive oxygen species. This study was designed to assess the effects of UDCA on oxidative injury and antioxidative systems in cultured rat hepatocytes. The viability of the hepatocytes dose-dependently decreased after hydrogen peroxide or cadmium administration. Pretreatment with UDCA significantly prevented this decrease in viability. The amounts of glutathione (GSH) and protein thiol increased significantly, but the activities of antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and catalase were unchanged in UDCA-treated hepatocytes. The mRNA levels of gamma-glutamylcysteine synthetase and metallothionein (MT) were significantly higher in UDCA-treated hepatocytes than in controls. In conclusion, UDCA increased hepatocyte levels of GSH and thiol-containing proteins such as MT, thereby protecting hepatocytes against oxidative injury. Our results provide a new perspective on the hepatoprotective effect of UDCA.