Anti-parkinsonian agents possessing both D₂ and D₃ receptor agonist properties are neuroprotective against 1-methyl-4-phenylpyridinium (MPP⁺) toxicity in a variety of in vitro models. The mechanisms underlying protection by these D₂/D₃ receptor agonists remain poorly defined. To test if the D₃ receptor preferring agonists S32504 and pramipexole act through D₂ or D₃ receptors and via brain-derived neurotrophic factor (BDNF)-dependent pathways, we utilized a terminally differentiated neuroblastoma SH-SY5Y cell line exhibiting a dopaminergic phenotype. The cytotoxic effects of MPP⁺ (LD₅₀ of 100 μM) were stereospecifically antagonized by S32504 (EC₅₀ = 2.0 μM) and, less potently, by pramipexole (EC₅₀ = 64.3 μM), but not by their inactive stereoisomers, R(+) pramipexole and S32601, respectively. Neuroprotective effects afforded by EC₅₀ doses of S32504 and pramipexole were antagonized by the selective D₃ antagonists S33084, U99194A, and SB269652, and by the D₂/D₃ antagonist raclopride. However, the preferential D₂ receptor antagonist LY741626 was ineffective as was the D1 antagonist SCH23390. BDNF (1 nM) potently protected against MPP⁺-induced neurotoxicity. Antibody directed against BDNF concentration-dependently blocked both the neuroprotective effects of BDNF and those of pramipexole and S32504 against MPP⁺. The protection afforded by BDNF was blocked by the P3K-AKT pathway inhibitor LY249002 and less so by the MEK/MAPKK pathway inhibitor PD98059. LY249002, but not PD98059, blocked the neuroprotective effects of pramipexole and S32504 against MPP⁺ toxicity. In conclusion, S32504 and, less potently, pramipexole show robust, stereospecific, and long-lasting neuroprotective effects against MPP⁺ toxicity that involve D₃ receptors. Their actions also reflect downstream recruitment of BDNF and via a PK3-AKT pathway.