Phenylketonuria (PKU) caused by phenylalanine hydroxylase (PAH) deficiency leads to toxic accumulation of phenylalanine (Phe). PAH is predominantly expressed in liver and its activity requires a supply of tetrahydrobiopterin (BH₄) cofactor, but we propose that expression of a complete Phe hydroxylating system (PAH plus BH₄ synthetic enzymes) in skeletal muscle will lead to therapeutic reduction of blood Phe levels in Pah^enu2 mice, a model of human PKU. In order to test this hypothesis, we first developed transgenic Pah^enu2 mice that lack liver PAH activity but coexpress, in their skeletal muscle, PAH and guanosine triphosphate cyclohydrolase I (GTPCH). The latter is responsible for the committing enzymatic step in BH₄ biosynthesis. Despite sufficient muscle enzyme expression, these mice remained hyperphenylalaninemic, thereby suggesting that expression of additional BH₄ synthetic enzymes would be necessary. A recombinant triple-cistronic adeno-associated virus-2 (AAV2) pseudotype 1 vector expressing PAH along with GTPCH and 6-pyruvoyltetrahydrobiopterin synthase (PTPS), the next step in BH₄ synthesis, was generated. Injection of this vector into the gastrocnemius muscles of Pah^enu2 mice led to stable and long-term reduction of blood Phe and reversal of PKU-associated coat hypopigmentation. We propose that muscle-directed gene therapy will be a viable alternative treatment approach to PKU and other inborn errors of metabolism.