We have analysed the standard deviation (SD) of blood phe and the dietary phe tolerance in 84 patients with PKU on dietary treatment during their first 4 years. Phe SD ranged from 70 to 460 μmol/l and phe intake from 120 to 940 mg/ day. There was a strong inverse correlation between phe SD and dietary phe tolerance (p <0.0001) confirming that those with 'severe' PKU showed a much higher variability in phe blood levels. Some individuals with a low phe SD but who, on the basis of their absolute blood phe levels had been assessed to need a relatively strict diet, were able to markedly increase their natural protein intake while still maintaining satisfactory phe control. We conclude that phe SD may be useful to provide additional information as to the severity of PKU and may suggest which patients might have a higher phe tolerance than previously thought. Missense mutations in the phenylalanine hydroxylase (PAH) gene can induce protein misfolding with loss of function of the PAH protein, the molecular basis of phenylketonuria. Sapropterin dihydrochloride, the synthetic form of tetrahydrobiopterin (BH4), was recently approved as the first pharmacological chaperone drug and shown to correct the loss-offunction phenotype by rescue of PAH misfolding. The aim of this study was to investigate the structure-function relationship of the BH4-molecule with respect to stabilization of PAH conformation. We compared the efficacy of BH4 and BH4-derivatives (BH2, Sepiapterin and 6-MPH4) with substitutions at the pterin ring system and/or the dihydroxypropyl side-chain to correct misfolding of wild-type and mutant PAH, respectively. BH4 and two dihydropterins (BH2, Sepiapterin) induced a compacted PAH conformation leading to mutation-specific increased thermal and thermodynamic stability. 6-MPH4, a cofactor without inhibitory potency lacking the dihydroxypropyl side-chain, led to protein destabilization. Interestingly, the presence of BH4 and BH2 but not Sepiapterin shifted the PAH unfolding model (2-state to 1-state). In conclusion, the structure-function relationship of these compounds is the molecular basis for structure aided drug design to improve the efficacy of pharmacological chaperones taking the patient's genotype into consideration.