Cytochrome P450 enzymes are responsible for the phase I metabolism of approximately 75% of known pharmaceuticals. P450s perform this and other important biological functions through the controlled activation of C-H bonds. Here, we report the spectroscopic and kinetic characterization of the long-sought principal intermediate involved in this process, P450 compound I (P450-I), which we prepared in approximately 75% yield by reacting ferric CYP119 with m-chloroperbenzoic acid. The Mössbauer spectrum of CYP119-I is similar to that of chloroperoxidase compound I, although its electron paramagnetic resonance spectrum reflects an increase in |J|/D, the ratio of the exchange coupling to the zero-field splitting. CYP119-I hydroxylates the unactivated C-H bonds of lauric acid [D(C-H) ~ 100 kilocalories per mole], with an apparent second-order rate constant of k(app) = 1.1 × 10(7) per molar per second at 4°C. Direct measurements put a lower limit of k ≥ 210 per second on the rate constant for bound substrate oxidation, whereas analyses involving kinetic isotope effects predict a value in excess of 1400 per second.