Tetrahydrobiopterin (BH 4) is a required cofactor for the enzymatic activity of phenylalanine hydroxylase (PAH) and is synthesized de novo from GTP in several tissues. Heterologous expression of PAH in tissues other than liver is a potential novel therapy for human phenylketonuria that is completely dependent upon BH 4 supply in the PAH-expressing tissue. Previous experiments with liver PAH-deficient transgenic mice that expressed PAH in skeletal muscle demonstrated transient correction of hyperphenylalaninemia only with hourly parenteral BH 4 administration. In this report, the fate of intravenously administered BH 4 is examined. The conclusions are that (1) BH 4 administered intravenously is rapidly taken up by liver and kidney, and (2) uptake of BH 4 into muscle is relatively low. The levels of BH 4 achieved in skeletal muscle following IV injection are only 10% of the amount expected were BH 4 freely and equally distributed across all tissues. The half-life of BH 4 in muscle is approximately 30 min, necessitating repeated injections to maintain muscle BH 4 content sufficient to support phenylalanine hydroxylation. The efficacy of heterologous muscle-directed gene therapy for the treatment of PKU will likely be limited by the BH 4 supply in PAH-expressing muscle.