Hepatic microsomes from rats pretreated with PCB were found to metabolize the food mutagen 2-amino-l-methyl-6-phenylimidazo(4, 5-b>)pyridine (PhIP) to two major metabolites, one of which was identified as the N-hydroxy derivative, 2-hydroxy-amino-l-methyl-6-phenylimidazo(4, 5-b)pyridine (N-OH-PhIP). This identification was based on mass spectral (MS), UV and HPLC data by comparison with N-OH-PhIP prepared by chemical synthesis, as well as the specific activity of the compound in the Ames Salmonella test. Synthetic N-OH-PhIP was prepared by catalytic reduction of the nitro derivative of PhIP, which was synthesized from PhIP by diazotization and reaction with sodium nitrite. N-OH-PhIP was mutagenic to Salmonella typhimurium TA98 without metabolic activation and had a specific mutagenic activity of 2700 revertants/nmol. N-OH-PhIP thus seems to be a proximate mutagenic metabolite of PhIP. Other direct acting mutagens were not detected in the microsomal incubation mixture after HPLC separation. N-OH-PhIP also induced sister chromatid exchange (SCE) in Chinese hamster ovary cells (CHO cells) without metabolic activation. The specific activity of N-OH-PhIP in this assay was ∼ 3 times higher than the activity of PhIP with microsomal activation.