The mechanisms by which hydrophobic bile acids are toxic to the liver are unknown. To determine whether the generation of free radicals is involved in the hepatotoxicity of bile acids, freshly isolated rat hepatocytes were incubated with individual bile acids (100 to 200 mumol/L) for 4 hr. Hepatocyte viability (trypan blue exclusion) declined to 40% to 50% in incubations with taurochenodeoxycholic acid and taurolithocholic acid, whereas taurocholic acid and tauroursodeoxycholic acid were not toxic. Lipid peroxidation was significantly associated with the loss of cell viability. Preincubation with different antioxidants-D-alpha-tocopheryl succinate, D-alpha-tocopherol, diphenyl-p-phenylenediamine, superoxide dismutase, catalase, superoxide dismutase + catalase, deferoxamine or apotransferrin-protected against the loss of viability and inhibited lipid peroxidation in cells incubated with 200 mumol/L taurolithocholic acid. alpha-Tocopheryl succinate added after 90 min of incubation with taurolithocholic acid ameliorated further hepatocyte toxicity and lipid peroxidation. Incubation of hepatocytes with 500 mumol/L of taurochenodeoxycholic acid or taurolithocholic acid under a low oxygen tension (9% O2) similarly caused lipid peroxidation and cell injury that was reversed by preincubation with D-alpha-tocopherol. These data suggest that oxygen free radicals may be involved in the pathogenesis of bile acid hepatotoxicity.