Tyrosine hydroxylase, a member of the aromatic amino acid hydroxylase family, uses a mononuclear Fe(H) and tetrahydropterin for hydroxylation of tyrosine to dihydroxyphenylalanine. Rapid-freeze quench Mossbauer spectroscopy has now provided direct evidence for the presence of an Fe(IV) intermediate in the reaction catalyzed by tyrosine hydroxylase. Rapid-quench techniques provide support for the kinetic competence of this species as the hydroxylating intermediate. This is the first direct evidence for a mononuclear Fe(IV) intermediate in an enzymatic aromatic hydroxylation reaction.