alpha-Synuclein is an abundant presynaptic protein implicated in neuronal plasticity and neurodegeneration disorders. Understanding alpha-synuclein function in dopaminergic cells could add to our knowledge of this key protein which is implicated in Parkinson's disease. Chronic or intermittent amphetamine (AMPH) abuse may create temporary or permanent disturbances in the dopaminergic system of the brain that may predispose individuals to Parkinsonism. Our previous studies showed that neurotoxicity induced by AMPH was mediated by enhanced oxidative stress and these effects were abolished by melatonin, a main secretory product of pineal gland. The present study was conducted to investigate the effect of AMPH on alpha-synuclein in regulating tyrosine hydroxylase (TH), a rate limiting enzyme for dopamine synthesis, in cultured human dopaminergic SK-N-SH cells. Of these, phosphorylation of Ser40 (pSer40) contributes significantly to TH activation and dopamine synthesis. Our data indicated that AMPH significantly increased the level of alpha-synuclein to 183% of the control value while reducing the levels of phosphorylated TH (TH-pSer40) enzyme and mitochondrial complex I to 78 and 52.9% of the control values, respectively and these effects were attenuated by melatonin. Further studies are needed to explore the mechanism by which alpha-synuclein contributes to TH-pSer40 dephosphorylation and the mechanism by which melatonin contributes to this interaction.