Aromatic L-amino acid decarboxylase (AADC) is necessary for conversion of L-DOPA to dopamine. Therefore, AADC gene therapy has been proposed to enhance pharmacological or gene therapies delivering L-DOPA. However, addition of AADC to the grafts of genetically modified cells expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1), which produce L-DOPA in parkinsonian rats, resulted in decreased production of L-DOPA and dopamine owing to feedback inhibition of TH by dopamine. End-product feedback inhibition has been shown to be mediated by the regulatory domain of TH, and site-specific mutation of serine 40 makes TH less susceptible to dopamine inhibition. Therefore, we investigated the efficacy of using TH with serine 40 mutated to leucine (mTH) in an ex vivo gene-therapy paradigm. Primary fibroblasts (PF) from Fischer 344 rats were transduced with retrovirus to express mTH or wild-type rat TH cDNA (wtTH). Both cell types were also transduced with GCH1 to provide the obligate TH cofactor, tetrahydrobiopterin. PF transfected with AADC were used as coculture and cografting partners. TH activities and L-DOPA production in culture were comparable between PFwtTHGC and PFmTHGC cells. In cocultures with PFAADC cells, PFmTHGC cells showed significant reduction in the inhibitory effect of dopamine compared with PFwtTHGC cells. In vivo microdialysis measurement showed that cografting PFAADC cells with PFmTHGC cells resulted in smaller decreases in L-DOPA and no reduction in dopamine levels compared with cografts of PFAADC cells with PFwtTHGC cells, which decreased both L-DOPA and dopamine levels. Maintenance of dopamine levels with lower levels of L-DOPA would result in more focused local delivery of dopamine and less potential side-effects arising from L-DOPA diffusion into other structures. These data support the hypothesis that mutation of serine 40 attenuates TH end-product inhibition in vivo and illustrates the importance of careful consideration of biochemical pathways and interactions between multiple genes in gene therapy.