There is increasing evidence that elevated production of reactive oxygen intermediates may contribute to the hepatotoxic actions of ethanol. Microsomes from ethanol-treated rats have been shown to generate oxygen radicals at elevated rates. Most of these studies have utilized chemical analysis techniques to assay for the production of reactive oxygen intermediates. Experiments employing the spin-trapping ESR spectroscopy technique were carried out for a more definitive characterization of production of reactive oxygen intermediates such as superoxide and hydroxyl radical (·OH) by microsomes from ethanol-fed rats and pair-fed controls, in the presence of either NADPH or NADH as the microsomal reductant. Superoxide production was determined by assaying the superoxide dismutase-sensitive generation of stable nitroxyl radical from 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine resulting from hydrogen abstraction by superoxide radical, while ·OH production was determined by assaying the formation of the POBN-α-hydroxyethyl radical adduct resulting from addition of the hydroxyethyl radical produced by hydrogen abstraction by ·OH. Microsomes from ethanol-treated rats displayed elevated rates of superoxide production with NADPH (50%) as well as with NADH (34%). With both microsomal preparations, the NADPH-dependent rates were three- to fourfold greater than the NADH-dependent rates. Microsomes from the ethanol-treated rats also displayed elevated rates of ·OH production (POBN-HER adduct formation) with NADPH (74%) and NADH (52%). With both microsomal preparations, NADPH-dependent rates were equivalent to the rates with NADH. The increase in superoxide and ·OH production after ethanol treatment was highly significant (n = 6, P < 0.001). Production of the POBN-HER adduct was sensitive to catalase, mannitol, and GSH, but not to superoxide dismutase. Addition of desferrioxamine caused a marked decrease in the signal intensity; the POBN-HER adduct formed under these conditions most likely reflects the previously reported radical-independent, direct oxidation of ethanol by cytochrome P450. Signal intensity in the presence of desferrioxamine was about threefold higher after ethanol treatment. These results demonstrate that production of reactive oxygen intermediates by microsomes, as determined by ESR spectroscopy, is elevated after chronic ethanol consumption, and that increases in production of reactive oxygen intermediates can be found with NADPH as well as NADH as the microsomal reductant.