Microsomes have the potential to oxidize alcohols by two pathways, one dependent on hydroxyl radicals (·OH) and the other dependent on cytochrome P-450 in which ·OH does not seem to be involved. The possibility that these two pathways may display differences in stereospecificity was evaluated by comparing the oxidation of (+)-2-butanol, (-)-2-butanol, and racemic 2-butanol. Microsomes oxidized 2-butanol to 2-butanone by a reaction which was partially sensitive to carbon monoxide and to competitive -OH scavengers. Desferrioxamine, which completely blocks the production of ·OH by microsomes, inhibited the oxidation of ethanol by about 60%, while the oxidation of 2-butanol and 1-butanol was decreased by only 30%. Vmax values for the oxidation of ethanol, 1-butanol, and 2-butanol were 17.7, 6.2, and 23.8 nmol min-1 (mg of protein), respectively, in the absence of desferrioxamine and 5.9, 4.7, and 13.6 nmol min-1 (mg of protein)-1, respectively, in the presence of desferrioxamine. 2-Butanol appears to be a particularly good alcohol substrate for the cytochrome P-450 dependent pathway of alcohol oxidation. Chronic ethanol consumption, which induces the microsomal alcohol oxidizing system, resulted in a 3-fold increase in the rate of 2-butanol oxidation. Most of this increment reflected an increased rate of metabolism by the cytochrome P-450 pathway. A type 2 binding spectrum was observed for the interaction of 2-butanol with microsomes from ethanol-fed rats, but not with controls. Hence, 2-butanol appears to be a very good alcohol substrate for the ethanol-inducible cytochrome P-450. The rates of oxidation of racemic 2-butanol, (+)-2-butanol, and (-)-2-butanol were the same for (a) model -OH-generating systems, (b) microsomes from chow-fed rats, and (c) microsomes from phenobarbital-treated rats. These results suggest that the ·OH-dependent and the cytochrome P-450 dependent pathways of alcohol oxidation by these microsomes do not display stereospecificity. By contrast, microsomes from the chronic ethanol-fed rats catalyzed the oxidation of the (+) enantiomer at rates twice that of the (-) enantiomer of 2-butanol. These differences were observed only for the cytochrome P-450 dependent pathway. Thus, the stereochemical preference displayed by microsomes from chronic alcohol fed rats may be due to the induction of a particular cytochrome P-450. Stereochemical studies may be of value in elucidating the mechanism and the identification of the ethanol-inducible cytochrome P-450.