The aim of this study was to determine the roles of reactive oxygen species (ROS), NF-ĸB and antioxidants in glycochenodeoxycholic acid (GCDC, 0–400 µmol/l, 0.5– 3 h)-induced hepatocytes death. The differential uptake of ethidium bromide and acridine orange revealed that apoptotic death occurred dose-dependently in GCDC-treated hepatocytes whereas necrotic death was prominent especially at higher GCDC concentrations (≧200 µmol/l). ROS generation measured fluorometrically either by a confocal laser microscope or by a microplate fluorescence reader was increased dose-dependently. The dose-dependent NF-ĸB activation with the significant IĸB-α decrease preceded both hepatocyte cell death and the alteration of antioxidant enzymes. The Cu/Zn-SOD level among several antioxidants, we checked, remained unchanged. In contrast, the catalase level and its enzymatic activity were markedly decreased only at 400 µmol/l. The Prx I and Prx II, newly defined antioxidant enzymes reducing H₂O₂ levels were decreased at the 200 and 400 µmol/l. These observations point to ROS generation in the GCDC-treated hepatocyte as the proximate event that triggers NF-ĸB activation, IĸB-α proteolysis, Prx depletion, and finally cell death. And oxidative stress may be more related to necrotic cell death in GCDC-treated hepatocytes.