Melatonin is a powerful antioxidant and free radical scavenger. A large body of in vivo and in vitro evidence shows that melatonin effectively inhibits membrane lipid peroxidation; this damage was based on the measurement of malondialdehyde and/or 4-hydroxynonenal levels. In the current study, for the first time using a more sensitive and specific biomarker, i.e. F2-isoprostanes, we investigate the effect of melatonin on diquat-induced lipid peroxidation in Fischer 344 rats. When diquat (40 mg/kg body weight) was intraperitoneally injected into rats, the levels of liver F2-isoprostanes were significantly increased at 1, 3, and 6 hr while plasma free F2-isoprostanes concentrations were augmented at 3, 6, and 12 hr after administration of the toxin. In addition, the plasma alanine aminotransferase activity level was measured as a parameter of hepatoxicity; the activity of this enzyme was augmented at 3, 6, and 12 hr after diquat administration when compared with levels of this constituent in untreated control rats. Pretreatment with melatonin (20 mg/kg) 30 min before diquat administration resulted in a significant reduction in both tissue and plasma F2-isoprostanes levels, and plasma alanine aminotransferase activity. These findings, using a sensitive and specific index of lipid peroxidation, show that the hepatoxicity of diquat, at least partially, is a consequence of reactive oxygen species-induced lipid damage. Melatonin's protective effects likely relate to its direct free radical scavenging ability and/or due to other antioxidative processes induced by the indole.