Metabolism of the heterocyclic amine carcinogen 2-amino-3-methylimidazo[4, 5-f]quinoline (IQ) was evaluated in mice with and without 40 mg/kg β-naphthoflavone (BNF). Following an oral dose of 40 mg/kg 14C-IQ, a 24-h urine sample was collected. Metabolism was assessed by high-performance liquid chromatography, and metabolites were identified by electrospray ionization mass spectrometry. Three new metabolites were identified as 1,2-dihydro-2-amino-5-hydroxy-3-methylimidazo[4,5-f]quinoline (m/z 217, [M + H]+), 1,2-dihydro-2-amino-5-O-glucuronide-3-methylimidazo[4,5-f] quinoline (m/z 393, [M + H]+), and 1,2-dihydro-2-amino-5,7-dihydroxy- 3-methylimidazo[4,5-f]quinoline (m/z 233, [M + H]+). These metabolites represented 21% of the total urinary radioactivity recovered. For BNF-treated mice, the abundance of metabolites observed was 5-O-glucuronide > m/z 217 > m/z 393 > 5-sulfate > m/z 233 > N-glucuronide > demethyl-IQ > sulfamate. In control mice, metabolite urinary abundance was 5-O-glucuronide > demethyl-IQ > sulfamate > N-glucuronide > m/z 217 > 5-sulfate. In liver slices from BNF-treated mice, synthesis of m/z 217 and 5-O-glucuronide was significantly reduced by ellipticine, a cytochrome P450 (P450) inhibitor, whereas sulfamate synthesis was significantly increased and demethyl-IQ was unchanged. Liver microsomes from BNF-treated mice produced m/z 217 and demethyl-IQ, with the former inhibited by ellipticine and furafylline, a selective 1A2 inhibitor, and the latter by ellipticine only. Injection (intraperitoneal) of demethyl-IQ into BNF-treated mice resulted in only a 30% conversion to three metabolites that were not observed in urine from animals receiving IQ. Results from BNF-treated mice showed significant IQ metabolism by hepatic P450s. Therefore, differences in metabolism between mice treated with and without BNF may affect IQ tumorigenicity.