Neonatal loading studies with tetrahydrobiopterin (BH 4 ) are used to detect hyperphenylalaninemia due to BH 4 deficiency by evaluating decreases in blood phenylalanine (Phe) concentrations post BH 4 load. BH 4 responsiveness in phenylalanine hydroxylase (PAH)-deficient patients introduced a new diagnostic aspect for this test. In older children, a broad spectrum of different levels of responsiveness has been described. The primary objective of this study was to develop a pharmacodynamic model to improve the description of individual sensitivity to BH 4 in the neonatal period. Secondary objectives were to evaluate BH 4 responsiveness in a large number of PAH-deficient patients from a neonatal screening program and in patients with various confirmed BH 4 deficiencies from the BIODEF database.Descriptive statistics in patients with PAH deficiency with 0–24-h data available showed that 129 of 340 patients (37.9%) had a >30% decrease in Phe levels post load. Patients with dihydropteridine reductase deficiency (n=53) could not be differentiated from BH 4 -responsive patients with PAH deficiency. The pharmacologic turnover model, “stimulation of loss” of Phe following BH 4 load, fitted the data best. Using the model, 193 of 194 (99.5%) patients with a proven BH 4 synthesis deficiency or recycling defect were classified as BH 4 sensitive. Among patients with PAH deficiency, 216 of 375 (57.6%) patients showed sensitivity to BH 4 , albeit with a pronounced variability; PAH-deficient patients with blood Phe <1200μmol/L at time 0 showed higher sensitivity than patients with blood Phe levels >1200μmol/L. External validation showed good correlation between the present approach, using 0–24-h blood Phe data, and the published 48-h prognostic test.Pharmacodynamic modeling of Phe levels following a BH 4 loading test is sufficiently powerful to detect a wide range of responsiveness, interpretable as a measure of sensitivity to BH 4 . However, the clinical relevance of small responses needs to be evaluated by further studies of their relationship to long-term response to BH 4 treatment.