Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and hypopigmentation of skin and hair. Low-phenylalanine diet therapy can prevent irreversible damage if instituted from birth. However, poor compliance with the strict lifelong dietary therapy leads to various neurologic and behavioral problems. To develop a safe and promising gene therapy method for PKU, we investigated whether a recombinant adeno-associated virus could be used as a PAH gene transfer vector to reduce the excessive phenylalanine level in the PKU mouse model. A recombinant adeno-associated virus vector encoding the human PAH gene (rAAV-hPAH), driven by EF1-alpha promoter, was infused into PAH-deficient mice, Pah(enu2), via the hepatic portal vein. Two weeks after injection, the plasma phenylalanine level dramatically decreased to 360 microM in male PKU mice, accompanied by the coat color changing to black. The mean plasma phenylalanine level of untreated PKU mice was 1800 microM. The PAH enzyme activities of treated mice increased to 10-17% of wild-type mice. No signs of liver toxicity were observed after gene transfer. The biochemical and phenotypic corrections were sustained for up to 25 wk (25-wk detection period). In contrast, the treatment was less effective in female PKU mice. These results indicate that recombinant adeno-associated virus vector-mediated gene therapy can be a useful therapeutic candidate for patients with PKU. Further studies are needed to clarify the differences in PKU pathogenesis in males and females, and to explore alternative administration routes besides hepatic portal vein injection.