OBJECTIVE Evidence is accumulating that hypoxic liver injury involves not only necrosis but also apoptosis. Reactive oxygen species can cause apoptosis. This study examined the hypothesis that H2O2 induces apoptosis in hypoxic rat liver. METHODS Blood-perfused rat livers were made hypoxic by reducing the perfusion flow. H2O2 was detected by both 2',7'-dichlorofluorescein fluoroimaging and cerium electron-microscopic methods. To evaluate the apoptosis, the liver was stained with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method. To further investigate the involvement of H2O2 in hypoxia-induced liver cell apoptosis, small pieces of liver in the cultured media were exposed to 0.5 mmol/L of reagent H2O2 and stained with the TUNEL method. RESULTS In the hypoxic liver, H2O2 was produced predominantly by hepatocytes, and the number of apoptotic nonparenchymal cells was significantly increased, particularly in the midzone. All the apoptotic cells were positively stained with monoclonal antibody against the hepatic sinusoidal endothelial cells (SECs). In incubated liver pieces, reagent H2O2 induced apoptosis selectively in SECs. CONCLUSIONS Low-flow hypoxia induces H2O2 production in hepatocytes, and this H2O2 induces apoptosis selectively in SECs in the rat liver.