Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme−57Fe(II)−phenylalanine−6-methyltetrahydropterin complex with O2 reveal the accumulation of an intermediate at short reaction times (20−100 ms). The Mössbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔEQ| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme−substrate complex reacts with oxygen to form an Fe(IV)═O intermediate with a rate constant of 19 mM−1 s−1, the Fe(IV)═O intermediate hydroxylates phenylalanine with a rate constant of 42 s−1, and rate-limiting product release occurs with a rate constant of 6 s−1 at 5 °C.