The influence of oxidative stress by hydrogen peroxide (H₂O₂) was examined in mouse primary cultured hepatocytes. A change in morphology was observed in hepatocytes incubated for 30min in saline A containing H₂O₂. The percentage of dead cells, as measured by the fluorescence method, was increased in a dose-dependent manner. In addition, a ladder-like DNA fragmentation pattern was detected by agarose gel electrophoresis 1 h after exposure to 3 mM H₂O₂. This phenomenon was prolonged for 24 h. Hydrogen peroxide-induced cell viability reduction and DNA fragmentation were dose-dependently protected by the addtion of antioxidants (N-acetylcysteine, L-ascorbic aicd), a metal-chelator (1, 10-phenanthroline), iron-chelator (deferoxamine) and intracellular calcium ion chelator (quin 2-AM). No influence, however, was detected by endonuclease inhibitors (zinc, aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitors (3-aminobenzamide, theophylline). These results following H₂O₂-induced cell viability reduction suggested that oxidative stress by H₂O₂ itself or H₂O₂-derived changes involved in ferrous or intracellular calcium ions resulted in apoptosis in mouse primary cultured hepatocytes. These phenomena are not likely to be associated with endonuclease or poly (ADP-ribose) polymerase.