Metabolism of heterocyclic amines to N-hydroxy intermediates appears critical in the mutagenic and carcinogenic actions of these compounds. We have studied the murine hepatic microsomal and cytosolic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine mutagen formed in cooked meats. PhIP (25 microM) was almost completely converted to N-hydroxy-PhIP and 4'-hydroxy-PhIP in 30 min by reaction with 3-methylcholanthrene-induced microsomal preparations. Microsomal formation of the active N-hydroxy-PhIP metabolite was slightly favored over the 4'-hydroxy-PhIP detoxification product at all concentrations studied (25-200 microM). Metabolism of PhIP in microsomal preparations derived from control mice was approximately 10% of the induced preparations. Metabolically activated PhIP and synthetic N-hydroxy-PhIP produced concentration-dependent increases in mutagenic activity in both Salmonella strains TA98 and TA98/1,8-DNP6, indicating that acetylated intermediates were not important in the mutagenicity of N-hydroxy-PhIP in these bacteria. Significant stabilization of the N-hydroxy-PhIP intermediate by both microsomal protein and BSA was observed. Addition of cytosol to microsomal incubations with PhIP (25 microM) resulted in an increase in mutagenic activity which could be attributable to stabilization by glutathione. An additional increase in mutagenicity resulted from addition of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), but not acetyl coenzyme A to microsomal preparations containing the cytosolic fraction. Furthermore, addition of PAPS to cytosolic preparations containing synthetic N-hydroxy-PhIP produced a 17% decrease in levels of the arylhydroxylamine relative to controls over 30 min, suggesting that secondary metabolism of N-hydroxy-PhIP to a sulfate conjugate may be relevant to the mutagenic and carcinogenic actions of PhIP.