Guanosine triphosphate (GTP) cyclohydrolase I, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH 4 ), is subject to feedback inhibition by BH 4 , a cofactor for phenylalanine hydroxylase. Inhibition was found to depend specifically on BH 4 and the presence of another protein (p35). The inhibition occurred through BH 4 -dependent complex formation between p35 protein and GTP cyclohydrolase I. Furthermore, the inhibition was specifically reversed by phenylalanine, and, in conjunction with p35, phenylalanine reduced the cooperativity of GTP cyclohydrolase I. These findings also provide a molecular basis for high plasma BH 4 concentrations observed in patients with hyperphenylalaninemia caused by phenylalanine hydroxylase deficiency.