The effect of iron and other compounds known to be toxic because of the production of oxygen radicals, e.g., paraquat and menadione on the generation of hydroxyl radicals (.OH) by microsomes from chronic ethanol-fed rats and their pair-fed controls was determined. In the absence of any additions, or in the presence of ferric-chloride, -ADP or -EDTA, microsomes from the ethanol-fed rats showed a 2-fold increase in the production of .OH. Paraquat and menadione increased the generation of .OH by microsomes from the ethanol-fed and the pair-fed controls to an identical extent and thus these promoters of oxidative stress were not any more effective in interacting with microsomes after ethanol treatment. Under all conditions, .OH generation was sensitive to inhibition by catalase, implicating H2O2 as the precursor of .OH, whereas superoxide dismutase was without any significant effect. A working scheme to accommodate aspects of the interaction of iron, menadione and paraquat with microsomes with the subsequent production of .OH is described. The fact that .OH generation by microsomes in the presence of several sources of iron such as unchelated iron or ferric-ADP is elevated after chronic ethanol consumption could contribute to the hepatotoxic effects of ethanol. Studies on iron metabolism by liver cells and the effect of ethanol on the disposition of this critical trace metal are needed to further evaluate the role of oxygen radicals in the actions of ethanol.