The oxidation of ethanol by rat liver microsomes is increased after chronic ethanol consumption. Previous experiments indicated that hydroxyl radicals play a role in the mechanism whereby microsomes oxidize ethanol. Experiments were therefore carried out to evaluate the role of these radicals in ethanol oxidation by microsomes from ethanol-fed rats, and to determine whether the increase in ethanol oxidation by these induced microsomes correlates with an increase in the generation of hydroxyl radicals. Rat liver microsomes from ethanol-fed rats catalyzed the oxidation of two typical hydroxyl radical scavenging agents, dimethylsulfoxide and 2-keto-4-thiomethylbutyric acid, at rates which were two- to threefold greater than rates found with control microsomes. This increased rate of oxidation of hydroxyl radical scavengers was similar to the increased rate of microsomal oxidation of ethanol. Azide, which inhibits contaminating catalase in microsomes, increased the oxidation of dimethyl sulfoxide and 2-keto-4-thiomethylbutyric acid by both microsomal preparations. This suggests that H2O2 may serve as the microsomal precursor of the hydroxyl radical. Cross competition for oxidation between ethanol and the hydroxyl radical scavenging agents was observed. Moreover, the oxidation of ethanol, dimethyl sulfoxide, or 2-keto-4-thiomethylbutyric acid was inhibited by other compounds which interact with hydroxyl radicals, e.g., benzoate, and the free-radical, spin-trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide. These results suggest that the increase in the rate of ethanol oxidation found with microsomes from ethanol-fed rats may be due, at least in part, to an increase in the rate of production of hydroxyl radicals by these induced microsomes. Increased production of oxyradicals may possibly result in oxidative damage to the liver cell as a result of ethanol consumption.