1. The hepatic microsomal metabolism of N-phenyl-2-naphthylamine (P2NA), an industrial antioxidant and suspected carcinogen, was studied in seven mammalian species to determine if N-dephenylation, reported to occur in vivo, could be demonstrated in vitro and if macromolecular binding occurs without an obligatory N-dephenylation. 2. The rate of hepatic microsomal metabolism decreased in the order: hamster much greater than mouse greater than rat congruent to monkey congruent to dog greater than human congruent to pig. Metabolism was increased by pretreatment with 3-methycholanthrene or phenobarbital and decreased by carbon monoxide or 2-[2,4-dichloro-6-phenyl)phenoxy]-ethylamine, which indicated the involvement of cytochrome P-450 rather than the flavoprotein mixed-function oxidase. 3. All seven species produced two major metabolites identified as 6-hydroxy-P2NA and 4-'hydroxy-P2NA. The carcinogen 2-naphthylamine was not detected in the microsomal incubations. 4. Incubations in the presence of 18O2 indicated that the oxygen incorporated in the products came from molecular oxygen. 5. There was a time-dependent linear increase in covalent binding of P2NA to microsomal protein and the extent of binding approximately paralleled the rate of metabolism. Thus, macromolecular binding of this aromatic amine appears to occur without an obligatory N-dephenylation and may be due to the metabolic formation of epoxides.