Rat liver microsomes generate a potent oxidant during electron-transfer reactions initiated by nicotinamide adenine dinucleotide phosphate (NADPH). The oxidant species exhibits the oxidizing power of the hydroxyl radical (·OH) and appears to be formed from H2O2 as precursor. Although a minor pathway of ethanol oxidation compared to alcohol dehydrogenase, the microsomal system assumes greater significance at elevated concentrations of ethanol and is inducible by the chronic consumption of ethanol. Some characteristics and the significance of the microsomal ethanol-oxidizing pathway are described. The mechanism at the molecular level for the oxidation of ethanol by liver microsomes is not clear. Ethanol can react with ·OH to produce acetaldehyde. Ethanol was oxidized by ·OH generated from model systems. Its oxidation by liver microsomes was inhibited by a series of competitive ·OH scavengers. Typical ·OH scavengers were oxidized during NADPH-dependent electron transfer. This chapter describes techniques for assaying the generation of ·OH-like oxidant species by microsomes and for evaluating its role in microsomal ethanol oxidation.