BACKGROUND: Tetrahydrobiopterin (BH(4)) is a potential new orphan drug for the treatment of some patients with phenylketonuria (PKU), mostly mild forms. Numerous studies have confirmed this finding and BH(4)-responsiveness may be predicted to some extent from the corresponding genotype. AIM: To investigate the response to BH(4) loading test, the phenylalanine hydroxylase (PAH) mutations and the long-term therapeutic efficacy of BH(4) in patients with PKU, and to better define BH(4)-responsive patients according to phenylalanine (Phe) levels and dietary phenylalanine tolerance. METHODS: 30 Italian PKU patients (age range: 6 months-24 years; 12 female, 18 male) were included in this retrospective study. Eleven out of 30 patients presented with Phe levels below 450 micromol/L and 19 patients with Phe levels between 450 and 900 micromol/L. In the second group, we investigated the effect of long-term (6 months-7 years) oral administration of BH(4) on blood Phe levels and daily Phe tolerance. RESULTS: In all patients with initial blood Phe levels <450 micromol/L (n = 11), BH(4) loading test was positive, but no treatment was introduced. In 12 out of 19 patients with blood Phe levels >450 micromol/L and positive at BH(4) loading, the treatment with BH(4) (10 mg/kg per day) was initiated. Before BH(4) treatment, Phe tolerance was less than 700 mg/day in all patients except for one (patient no. 9), increasing to 2-3-fold (from 498 +/- 49 to 1475 +/- 155 mg/day) on BH(4) treatment. In these patients the amino acid mixture supplementation was stopped and the diet was a combination of low-protein foods and natural proteins, mostly from animal sources. CONCLUSION: Long-term BH(4) substitution (up to 7 years) in a group of moderate PKU patients allowed a substantial relaxation of the dietary restrictions or even replacement of the diet with BH(4) without any adverse effects.