BH(4) administration results in the reduction of blood phenylalanine level in patients with tetrahydrobiopterin (BH(4))-responsive phenylalanine hydroxylase (PAH) deficiency. The mechanism underlying BH(4) response remains unknown. Here, we studied the effects of BH(4) and phenylalanine on in vivo PAH activity of normal controls using the phenylalanine breath test (PBT) by converting l-[1-(13)C] phenylalanine to (13)CO(2). Phenylalanine oxidation rates were expressed as Delta(13)C ((13)CO(2)/(12+13)CO(2), per thousand) and cumulative recovery rates over 120min (CRR(120), %; total amount of (13)CO(2)/the administered dose of (13)C-phenylalanine). Under physiological conditions of blood phenylalanine, BH(4) administration reduced the Delta(13)C peak from 40.8 per thousand to 21.6 per thousand and CRR(120) from 16.9% to 10.2%. Under high blood phenylalanine conditions, administration of BH(4) increased the Delta(13)C peak from 30.7 per thousand to 46.0 per thousand, while the CRR(120) was similar between phenylalanine (19.9%) and phenylalanine+BH(4) (21.1%) groups. Corrected Delta(13)C and CRR(120) were calculated against serum phenylalanine levels to remove the effects of phenylalanine loading. After BH(4) administration, the corrected Delta(13)C peak increased from 82.7 per thousand to 112.6 per thousand, while the corrected CRR(120) was similar (47.6% and 45.6%). These results indicate that phenylalanine worked as a regulator of in vivo PAH by serving as both a substrate and an activator for the enzyme. Excessive dosages of BH(4) inhibited PAH under normal phenylalanine conditions and activated PAH under conditions of high phenylalanine. The regulation system is therefore designed to maintain phenylalanine levels in the human body. Appropriate BH(4) supplementation must be reviewed in patients with BH(4)-responsive PAH deficiency.