2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant compound of the amino-imidazoazaarens (AIA) group of muta-/carcinogens isolated from the crust of fried meat. PhIP is principally activated via P450IA2 dependent N2-hydroxylation. A major metabolic pathway is N2-glucuronidation of the proximate 2-hydroxyamino-PhIP metabolite and excretion via bile to the intestine. After bacterial hydrolysis the proximate metabolite may be esterified by the intestinal cells and cause genetic damage. 2-Hydroxyamino-PhIP formed in vivo may be further oxidized presumably to 2-nitro-PhIP which reacts directly with glutathione through substitution of the nitro group. Detoxification is principally via P450IA1 dependent ring-hydroxylation followed by sulfation or glucuronidation. Direct glucuronidation also occurs. PhIP metabolism was examined in freshly isolated hepatocytes from rat, mouse, hamster and guinea pig. Activation was evaluated by the total level of covalent binding of PhIP to macromolecules. Rat hepatocytes had the lowest rate of metabolism, both to reactive and detoxified metabolites. The major products were 4'PhIP-sulfate, PhIP-glucuronide and 2-hydroxyamino-PhIP glucuronide, whereas in the mouse hepatocytes mainly 4'PhIP-sulfate was found. The level of covalent binding in the mouse hepatocytes exceeded those of the rat. An extensive metabolism was seen in guinea pig hepatocytes, the major products being 4'PhIP-sulfate, 4'-O-PhIP glucuronide, PhIP-glucuronide and 2-hydroxyamino-PhIP-glucuronide. The relative amount of PhIP covalently bound to macromolecules in guinea pig hepatocytes was low. Hamster hepatocytes had the highest level of covalently bound PhIP. The main metabolites were 4'PhIP-sulfate, 4'O-PhIP-glucuronide and PhIP-glucuronide. Minor amounts of 2-hydroxyamino-PhIP-glucuronide was produced in the hamster. Several unknown PhIP metabolites were formed in the hamster and guinea pig. Direct detoxification of PhIP and further metabolism of 2-hydroxyamino-PhIP to reactive and/or detoxified metabolites are important for the resulting covalent binding.