The specificity of rabbit cytochrome P-450 involved in the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was assessed using control and induced rabbit liver and lung microsomes, and six purified forms of cytochrome P-450. The number of revertants produced/2.5 micrograms PhIP by control rabbit liver was 260 +/- 196/10 micrograms of microsomal protein (mean +/- SD; n = 3), and this increased to 1265 +/- 248 when 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced liver microsomes were used as the activation source in the Ames test. Microsomes form phenobarbital-, rifampicin- and acetone-pretreated rabbits showed no increase in activity over controls. Control lung microsomes did not activate PhIP to a mutagen, whereas TCDD-induced lung microsomes produced 1443 +/- 136 (mean +/- SD; n = 4) Ames/Salmonella revertants/100 micrograms protein. In reconstitution experiments cytochrome P450 forms 4 and 6 were found to be efficient activators of PhIP to a mutagen Form 6 was 3.1-fold more active than form 4 and produced 4577 revertants/10 pmol with a 20-min preincubation step in the Ames test. Cytochrome form 5 produced 17 revertants/10 pmol and forms 2, 3b and 3c were not active in metabolizing PhIP to a mutagen. A highly significant statistical correlation existed between the capacity of control and induced liver microsomes to activate PhIP to a mutagen and their cytochrome P-450 form 4 (r = 0.97, r2 = 0.94) and form 6 (r = 0.95, r2 = 0.90) content. These data strongly support the involvement of polycyclic hydrocarbon-inducible forms of cytochrome P450 in the activation of PhIP in the rabbit. Anti-rabbit forms 4 and 6 IgGs recognized proteins in seven human liver microsomes of comparable mol. wt to rabbit cytochrome P-450 forms 4 and 6. However, no correlation existed between the content of these proteins and the capacity of human liver microsomes to activate PhIP.