Styrene monooxygenase (SMO) is a two-component flavoenzyme composed of an NADH-specific flavin reductase (SMOB) and FAD-specific styrene epoxidase (NSMOA). NSMOA binds tightly to reduced FAD and catalyzes the stereospecific addition of one atom of molecular oxygen to the vinyl side chain of styrene in the enantioselective synthesis of S-styrene oxide. In this mechanism, molecular oxygen first reacts with NSMOA(FAD(red)) to yield an FAD C(4a)-peroxide intermediate. This species is nonfluorescent and has an absorbance maximum of 382 nm. Styrene then reacts with the peroxide intermediate with a second-order rate constant of (2.6 ± 0.1) × 10(6) M(-1) s(-1) to yield a fluorescent intermediate with an absorbance maximum of 368 nm. We compute an activation free energy of 8.7 kcal/mol for the oxygenation step, in good agreement with that expected for a peroxide-catalyzed epoxidation, and acid-quenched samples recovered at defined time points in the single-turnover reaction indicate that styrene oxide synthesis is coincident with the formation phase of the fluorescent intermediate. These findings support FAD C(4a)-peroxide being the oxygen atom donor and the identity of the fluorescent intermediate as an FAD C(4a)-hydroxide product of the styrene epoxidation. Overall, four pH-dependent rate constants corresponding to peroxyflavin formation (pK(a) = 7.2), styrene epoxidation (pK(a) = 7.7), styrene oxide dissociation (pK(a) = 8.3), and hydroxyflavin dehydration (pK(a) = 7.6) are needed to fit the single-turnover kinetics.