Some properties of the monooxygenase activity measured in the recently described dichloro- p-nitroanisole O-demethylase assay were investigated in rat liver microsomes. Enzyme activity in control microsomes was inhibited by ethanol (I 50 = 5 mM). The small slope of the inhibition curve was interpreted as evidence for a heterogenous enzyme system, with at least one enzyme being highly sensitive to ethanol inhibition. A number of other water-miscible organic solvents also inhibited the reaction, tetrahydrofuran being most efficient (I 50 = 0.6 mM). Tween 80 gave only weak inhibition at concentrations up to 4 mg/ml. Vinyl chloride was a strong inhibitor. Phenobarbital pretreatment of the rats increased O-demethylase activity at least 24-fold, and made the enzyme highly sensitive to metyrapone inhibition but not to ethanol. P-448 was not involved in the reaction, since 3-methylcholanthrene pretreatment, or addition of 7, 8-benzoflavone, produced no effect. Only small differences were found between the enzyme from male and female rats.