Viral vector-mediated gene transfer is emerging as a novel therapeutic approach with clinical utility in treatment of Parkinson's disease. Recombinant adeno-associated viral (rAAV) vector in particular has been utilized for continuous l-3,4 dihydroxyphenylalanine (DOPA) delivery by expressing the tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) genes which are necessary and sufficient for efficient synthesis of DOPA from dietary tyrosine. The present study was designed to determine the optimal stoichiometric relationship between TH and GCH1 genes for ectopic DOPA production and the cellular machinery involved in its synthesis, storage, and metabolism. For this purpose, we injected a fixed amount of rAAV5-TH vector and increasing amounts of rAAV5-GCH1 into the striatum of rats with complete unilateral dopamine lesion. After 7 weeks the animals were killed for either biochemical or histological analysis. We show that increasing the availability of 5,6,7,8-tetrahydro-l-biopterin (BH4) in the same cellular compartment as the TH enzyme resulted in better efficiency in DOPA synthesis, most likely by hindering inactivation of the enzyme and increasing its stability. Importantly, the BH4 synthesis from ectopic GCH1 expression was saturable, yielding optimal TH enzyme functionality between GCH1 : TH ratios of 1 : 3 and 1 : 7.