The principal rat liver microsomal metabolite of 4-nitroaniline was isolated by high pressure liquid chromatography and was characterized as 2-amino-5-nitrophenol (2-hydroxy-4-nitroaniline) by comparison of its mass, nuclear magnetic resonance, and ultraviolet spectra and HPLC retention time to the synthetic compound. A metabolite with the chromatographic retention time of authentic N-hydroxy-4-nitroaniline was not detected. Pretreatment of rats with phenobarbital and 3-methylcholanthrene increased the rate of conversion of 4-nitroaniline to 2-hydroxy-4-nitroaniline by 2-fold and 4-fold, respectively; the reaction required NADPH and was inhibited by heat treatment of microsomes, by argon and carbon monoxide:oxygen atmospheres and by the cytochrome P-450 inhibitor, 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine. In the presence of a molecular oxygen (18O2) atmosphere, 18O was quantitatively incorporated into the metabolite. Microsomes did not catalyze the isomerization of N-hydroxy-4-nitroaniline to 2-hydroxy-4-nitroaniline. A primary isotope effect was not observed upon comparison of the rate of conversion of 2,6-dideutero-4-nitroaniline to 2-hydroxy-4-nitroaniline with that of the nondeuterated compound. The 2-hydroxy-4-nitroaniline derived from microsomal incubation mixtures of 2,6-dideutero-4-nitroaniline contained about 20%, 3,6-dideutero-2-hydroxy-4-nitroaniline.