In humans, mutations in the α-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson’s disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. α-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves α-synuclein, we generated α-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting α-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the α-synuclein gene. As described previously, deletion of α-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, α-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the α-synuclein gene exhibited no protection. Furthermore, α-synuclein knockout mice but not the mice with the α-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that α-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.