The cofactor tetrahydrobiopterin deficiency, which is required for phenylalanine hydroxylase activity, leads to hyperphenylalaninemia. By blood phenylalanine levels elevation, loss of IQ, poor school performance, and behavior problems will occur. In GTP cyclohydrolase deficiency, very little tetrahydrobiopterin synthesis occurs and both neopterin and biopterin concentrations will be decrease. In this study blood specimens from 79 healthy volunteers and 83 patients with PKU were processed for measurement of GTP-cyclohydrolase activity and determination of pterins by chromatography. Our results showed that tetrahydrobiopterin in the some of PKU group was decreased compared to that in control [12.46±0.23, (mean±SD., mmol/L, n=83) vs. 68.23±0.74, n=79,P<0.05 J. GTP-cyclohydrolase activity in PKU group was also decreased compared to that in controls. Results of this study indicate that examination of pterins and GTP-cyclohydrolase activity are helpful and can lead to the diagnosis of hyperphenylalaninemia.