Recent analyses of autopsied brains from subjects previously administered AAV2-neurturin (NRTN) gene transfer argues that optimizing the effects of neurotrophic factors in Parkinson’s disease (PD) likely requires delivery to both the degenerating cell bodies (in substantia nigra) and their terminals (in striatum). Prior to implementing this novel dosing paradigm in humans, we conducted eight nonclinical experiments with three general objectives: (1) evaluate the feasibility, safety and effectiveness of targeting the substantia nigra (SN) with AAV2-NRTN, (2) better understand and appraise recent warnings of serious weight loss that might occur with targeting the SN with neurotrophic factors, and (3) define an appropriate dose of AAV2-NRTN that should safely and effectively cover the SN in PD patients. Toward these ends, we first determined SN volume for rats, monkeys and humans, and employed these values to calculate comparable dose equivalents for each species by scaling each dose, based on relative SN volume. Using this information, we next injected AAV2-GFP to monkey SN to quantify AAV2-vector distribution and confirm reasonable SN coverage. We then selected and administered a ~200-fold range of AAV2-NRTN doses (and a single AAV2-GDNF dose) to rat SN, producing a wide range of protein expression. In contrast to recent warnings regarding nigra targeting, no dose produced any serious side effects or toxicity, though we replicated the modest reduction in weight gain reported by others with the highest AAV2-NRTN and the AAV2-GDNF dose. A dose-related increase in NRTN expression was seen, with the lower doses limiting NRTN to the peri-SN and the highest dose producing mistargeted NRTN well outside the SN. We then demonstrated that the reduction in weight gain following excessive-doses can be dissociated from NRTN in the targeted SN, and is linked to mistargeted NRTN in the diencephalon. We also showed that prior destruction of the dopaminergic SN neurons via 6-OHDA had no impact on the weight loss phenomenon, further dissociating neurotrophic exposure to the SN as the culprit for weight changes. Finally, low AAV2-NRTN doses provided significant neuroprotection against 6-OHDA toxicity, establishing a wide therapeutic index for nigral targeting. These data support targeting the SN with AAV2-NRTN in PD patients, demonstrating that properly targeted and scaled AAV2-NRTN provides safe and effective NRTN expression. They also provided the means to define an appropriate human-equivalent dose for proceeding into an ongoing clinical trial, using empirically-based scaling to account for marked differences in SN volume between species.