A monoclonal anti-idiotype antibody, NS7, previously shown to mimic the binding of the pterin cofactor of phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been used to localize the cofactor binding site within the phenylalanine hydroxylase catalytic domain to a 27-amino-acid sequence that is highly conserved among the three aromatic amino acid hydroxylases. The binding of NS7 to a synthetic peptide corresponding to the phenylalanine hydroxylase sequence from residue 263 to residue 289 was blocked by the competitive inhibitor of phenylalanine hydroxylase enzyme activity, 7,8-dihydro-6,7-dimethylpterin. In addition this peptide competed with native phenylalanine hydroxylase for binding to 6,7-dimethyl-5,6,7,8-tetrahydropterin conjugated to a polyglutamate carrier. Application of this simple and direct approach to other enzymes is likely to greatly facilitate the identification of ligand binding sites on enzymes, which will significantly contribute to the understanding of enzyme structure-function relationships.