Analysis of repetitive scan difference spectra of incubation mixtures containing liver microsomes from phenobarbital-pretreated rats, benzo(a)pyrene-3,6-quinone, and NADPH reveals the rapid reduction of the quinone to a steady-state level of hydroquinone and the subsequent reoxidation of the hydroquinone. This cyclic process results in NADPH oxidation coupled to oxygen reduction and hydrogen peroxide formation. The reduction of the benzo(a)pyrene-3,6-quinone is not supported by NADH. The initial rate of the NADPH-supported reaction is inhibited by NADP +, metyrapone, and antiNADPH-cytochrome P-450 ( c ) reductase globulin, but not by dicumarol, anaerobiosis, or a gas mixture of carbon monoxide and oxygen (4:1, v v ). These results suggest that cytochrome P-450 and its reductase are involved in this reaction. During the steady-state of metabolism of benzo(a)pyrene by liver microsomes, the 3,6-quinone produced must exist largely as the hydroquinone and may be suitable for disposition as a water-soluble conjugate(s).