The 14-3-3 proteins stimulate the activation of tyrosine hydroxylase (TH), the rate-limiting catecholamine biosynthetic enzyme. To explore if particular endogenous 14-3-3 isoforms specifically affected TH activity and dopamine synthesis, we utilized rodent nigrostriatal tissues and midbrain-derived MN9D dopaminergic cells. Extracts from ventral midbrain and MN9D cells contained similar pools of 14-3-3 mRNAs, with 14-3-3zeta being relatively abundant in both. Protein levels of 14-3-3zeta were also abundant. [(32)P]Orthophosphate labeling of MN9D cells, followed by co-immunoprecipitation with pan-TH and pan-14-3-3 antibodies brought down similar amounts of phosphorylated TH in each, confirming that 14-3-3-bound phosphorylated TH in our cells. Co-immunoprecipitation of striatal tissues with a pan-TH antibody precipitated 14-3-3zeta but not another potential TH regulatory isoform, 14-3-3eta. In whole cell extracts from MN9D cells after 14-3-3 small interfering RNA treatments, we found that 14-3-3zeta knockdown significantly reduced TH activity and dopamine synthesis whereas knockdown of 14-3-3eta had no effect. 14-3-3zeta was found co-localized on mitochondria with TH, and its knockdown by small interfering RNA reduced TH phosphorylation and TH activity in the mitochondrial pool. Together the data support a role for 14-3-3zeta as an endogenous activator of TH in midbrain dopaminergic neurons and furthermore, identify mitochondria as a potential novel site for dopamine synthesis, with implications for Parkinson disease.