Background: Increasing gene dosages of α-synuclein induce familial Parkinson's disease (PD); thus, the hypothesis has been put forward that regulation of gene expression, in particular altered α-synuclein gene methylation, might be associated with sporadic PD and could be used as a biological marker. Methods: We performed a thorough analysis of α-synuclein methylation in bisulfite-treated DNA from peripheral blood of 490 sporadic PD patients and 485 healthy controls and in addition analyzed the effect of levodopa (l-dopa) on α-synuclein methylation and expression in cultured mononuclear cells. Results: α-Synuclein was hypomethylated in sporadic PD patients, correlated with sex, age, and a polymorphism in the analyzed sequence stretch (rs3756063). α-Synuclein methylation separated healthy individuals from sporadic PD with a specificity of 74% (male) and 78% (female), respectively. α-Synuclein methylation was increased in sporadic PD patients with higher l-dopa dosage, and l-dopa specifically induced methylation of α-synuclein intron 1 in cultured mononuclear cells. Conclusions: α-Synuclein methylation levels depended on disease status, sex, age, and the genotype of rs3756063. The pharmacological action of l-dopa was not limited to the dopamine precursor function but included epigenetic off-target effects. The hypomethylation of α-synuclein in sporadic PD patients' blood already observed in previous studies was probably underestimated because of effect of l-dopa, which was not known previously. The analysis of α-synuclein methylation can help to identify nonparkinsonian individuals with reasonable specificity, which offers a valuable tool for researchers and clinicians.