One major point of controversy in the area of cytochrome P450 (P450)-mediated oxidation reactions is the nature of the active-oxygen species. A number of hypotheses have been advanced which implicate a second oxidant besides the iron-oxo species designated as compound I (Cpd 1). This oxygen is thought to be either an iron-hydroperoxy species (Cpd 0) or a second spin-state of Cpd 1. Very little information is available on what fraction of P450 oxidations is mediated by the two different oxidants. Herein, we report results on three cytochrome P450-mediated reactions: O-dealkylation, N-oxygenation, and aromatic hydroxylation, which occur by three distinct chemical mechanisms. We have used kinetic isotope effects to test for branching from O-demethylation to N-oxygenation and aromatic hydroxylation, using 6-methoxyquinoline and 2H3-6-methoxyquinoline as substrates for P4501A2. Identical large inverse isotope effects on Vmax/Km are obtained for the formation of both the N-oxide and the phenol. This indicates that all three reactions occur through the same enzyme-substrate complex and, thus, through a single iron-oxygen species. The nature of the iron-oxygen species is less certain but is more likely to be iron-oxo Cpd 1, given the energetics of these reactions.