Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.