The pathogenetic role of lipid peroxidation in ethanol-induced liver injury was previously supported by demonstration of increased formation of diene conjugates and decreased hepatic levels of reduced glutathione in ethanol-fed animals and alcoholic patients with liver injury. The present study was carried out to investigate whether these findings can be extended to a rat model that was shown to produce a spontaneous ethanol-induced liver injury progressing from steatosis to necrosis and fibrosis (Hepatology 6: 814, 1986). Despite the histological evidence of progression from hepatic steatosis to centrilobular necrosis in these animals, diene conjugate formation in mitochondrial and microsomal lipids was not enhanced when compared to pair-fed controls. In addition, hepatic levels of neither methionine nor glutathione were decreased in the ethanol-fed animals. The fatty acid composition of mitochondrial phospholipids from these animals was similar to that in the controls. However, in the microsomal phospholipids, the level of arachidonate (20:4) was depressed by about 50% as compared to the controls. These results demonstrate the lack of evidence for a pathogenetic relationship between lipid peroxidation and ethanol-induced liver injury progressing to centrilobular necrosis. They further suggest that the decreased levels of 20:4 commonly seen after chronic ethanol intake may not be due to a peroxidative loss.