Naphthalene (NA) is metabolized by pulmonary and hepatic tissues to epoxides, quinones, and their related phase II metabolites. To delineate specific liver metabolites, metabolism was studied in B6C3F1 mouse liver perfused with 5 and 10 mumol/hr [14C]NA. Liver metabolites were compared with urinary metabolites from mice exposed to an equivalent total dose of NA (50 mg/kg ip) to ascertain interorgan and extrahepatic transformation in vivo. Metabolites were separated into pools via hydrophobic columns under neutral (pool I) and acid (pool II) conditions. Pool I contained the majority of [14C] in perfusate and urine. In perfusate, high levels of sulfate conjugates of naphthol and dihydroxynaphthalene were found along with dihydrodiol and glucuronic acid conjugates. In the urine, dihydrodiol was the most abundant metabolite. A novel N-acetylated glutathione conjugate was a constituent of pool II of both perfusate and urine. Additional metabolites identified in urine were N-acetylcysteine conjugate of dihydrodiol epoxide, mercaptolactic acid conjugate of naphthalene oxide (NO), and diglucuronide and sulfate/glucuronide conjugates of dihydroxynaphthalene. Mercapturic acid conjugates of NO were not observed in either perfusate or urine; this finding highlights metabolic differences between strains. Differential covalent binding occurred in cellular fractions, with the highest binding occurring in microsomes and mitochondria. These metabolites indicate that interorgan metabolism plays a role in the disposition of NA in vivo.