Density functional calculations were performed in response to the controversies regarding the identity of the oxidant species in cytochrome P450. The calculations were used to gauge the relative C-H hydroxylation reactivity of three potential oxidant species of the enzyme, the high-valent oxo-iron species Compound I (Cpd I), the ferric hydroperoxide Compound 0 (Cpd 0), and the ferric-hydrogen peroxide complex Fe(H(2)O(2)). The results for the hydroxylation of a radical probe substrate, 1, show the following trends: (a) Cpd I is the most reactive species; in its presence the other two reagents will be silent. (b) In the absence of Cpd I, substrate oxidation by Cpd 0 and Fe(H(2)O(2)) will take place via a stepwise mechanism that involves initial O-O homolysis followed by H-abstraction from 1. (c) Cpd 0 will undergo mostly porphyrin hydroxylation and only approximately 15% of substrate oxidation producing mostly the rearranged alcohol, 3 (Scheme 2). (d) Fe(H(2)O(2)) will generate mostly free hydrogen peroxide (uncoupling). A small fraction will perform substrate oxidation and lead mostly to 3. Reactivity probes for these reagents are kinetic isotope effect (KIE) and the product ratio of unrearranged to rearranged alcohols, [2/3]. Thus, for substrate oxidation by Cpd 0 or Fe(H(2)O(2)) KIE will be small, approximately 2, while Cpd I will have large KIE values. Typically both Cpd 0 and Fe(H(2)O(2)) will lead to a [2/3] ratio < 1, while Cpd I will lead to ratios > 1. In addition, the product isotope effect (KIE(2)/KIE(3) not equal 1) is expected from the reactivity of Cpd I.