Substitution of deuterium for hydrogen on the alpha-carbon of 7-ethoxycoumarin results in an intrinsic isotope effect of approximately 14 during the cytochrome P-448-catalyzed O-deethylation of this substrate (G. T. Miwa, J. S. Walsh, and A. Y. H. Lu (1984) J. Biol. Chem. 259, 3000-3004). This dramatic decrease in the C-H bond cleavage rate does not, however, alter the rate of 7-ethoxycoumarin disappearance or the rates of NADPH and oxygen consumption indicating that the catalytic turnover of this enzyme is unaffected. Moreover, hydrogen peroxide formation and the concentrations of an oxycytochrome P-448 complex (lambda max = 440 nm) are also unchanged demonstrating that the steady state concentrations of various oxy intermediates of cytochrome P-448 are also unchanged. An inescapable conclusion from these data is that an irreversible step exists between the formation of these intermediates and the oxidation of the substrate. These data are in agreement with the view that an irreversible cleavage of the dioxygen bond precedes substrate oxidation. Moreover, the oxidatively competent form of the cytochrome must then be committed to substrate oxidation. The latter conclusion is substantiated from high performance liquid chromatography studies which demonstrate the formation of a second metabolite, 6-hydroxy-7-ethoxycoumarin, from the deuterated substrate arising from the metabolic switching away from the O-ethyl group to the aromatic ring of this substrate.