Background Tyrosine hydroxylase (TH) regulates dopamine (DA) bioavailability. Its product, L-DOPA, is an established treatment for Parkinson's disease (PD), suggesting that TH regulation influences locomotion. Site-specific phosphorylation of TH at ser31 and ser40 regulates activity. No direct evidence shows that ser40 phosphorylation is the dominating mechanism of regulating TH activity in vivo, and physiologically-relevant stimuli increase L-DOPA biosynthesis independent of ser40 phosphorylation. Significant loss of locomotor activity occurs in aging as in PD, despite less loss of striatal DA or TH in aging compared to the loss associated with symptomatic PD. However, in the substantia nigra (SN), there is equivalent loss of DA or TH in aging and at the onset of PD symptoms. Growth factors increase locomotor activity in both PD and aging models and increase DA bioavailability and ser31 TH phosphorylation in SN, suggesting that ser31 TH phosphorylation status in the SN, not striatum, regulates DA bioavailability necessary for locomotor activity. Methodology and Principal Findings We longitudinally characterized locomotor activity in young and older Brown-Norway Fischer 344 F1 hybrid rats (18 months apart in age) at two time periods, eight months apart. The aged group served as an intact and pharmacologically-naïve source of deficient locomotor activity. Following locomotor testing, we analyzed DA tissue content, TH protein, and TH phosphorylation in striatum, SN, nucleus accumbens, and VTA. Levels of TH protein combined with ser31 phosphorylation alone reflected inherent differences in DA levels among the four regions. Measures strictly pertaining to locomotor activity initiation significantly correlated to DA content only in the SN. Nigral TH protein and ser31 phosphorylation together significantly correlated to test subject's maximum movement number, horizontal activity, and duration. Conclusions/Significance Together, these results show ser31 TH phosphorylation regulates DA bioavailability in intact neuropil, its status in the SN may regulate locomotor activity generation, and it may represent an accurate target for treating locomotor deficiency. They also show that neurotransmitter regulation in cell body regions can mediate behavioral outcomes and that ser31 TH phosphorylation plays a role in behaviors dependent upon catecholamines, such as dopamine.