Oxidant stress is critically involved in various liver diseases. Superoxide formation causes c-jun N-terminal kinase (JNK)- and caspase-dependent apoptosis in cultured hepatocytes. To verify these findings in vivo, male Fisher rats were treated with diquat and menadione. The oxidant stress induced by both compounds was confirmed by increased formation of glutathione disulfide and 4-hydroxynonenal protein adducts. Plasma ALT activities increased from 46±4 U/L in controls to 955±90 U/L at 6 h after diquat treatment. H&E staining of liver sections revealed large areas of necrotic cells at 3 and 6h. DNA strandbreaks, evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, showed clusters of TUNEL-positive cells, where the staining was predominantly cytosolic and the cells were swollen indicating oncotic necrosis. There was no significant increase in caspase-3 activities or relevant release of DNA fragments into the cytosol at any time between 0 and 6h after diquat treatment. Despite the activation of JNK after high doses of diquat, the JNK inhibitor SP600125 did not protect against diquat-induced necrosis. Menadione alone did not cause liver injury but in combination with phorone and FeSO₄ induced moderate oncotic necrosis. On the other hand, if animals were treated with galactosamine/endotoxin as positive control for apoptosis, caspase-3 activities were increased by 259%, the number of TUNEL-positive cells with apoptotic morphology was increased 103-fold and DNA fragmentation was enhanced 6-fold. The data indicate that liver cell death initiated by diquat-induced superoxide formation in vivo is mediated predominantly by oncotic necrosis and is independent of JNK activation.