Parkinson's disease is characterized by progressive neuronal degeneration of dopaminergic neurons in the substantia nigra. Many factors are thought to contribute to the neuronal cell death that occurs in Parkinson's disease, including alpha-synuclein-mediated toxicity. Previously, we have reported that alpha-synuclein directly couples to the carboxyl tail of the dopamine transporter (DAT) and that the alpha-synuclein/DAT protein complex formation accelerates DAT-mediated cellular dopamine (DA) uptake and DA-induced cellular apoptosis. In the present study, we report that parkin, an E2-dependent E3 protein ubiquitin ligase associated with recessive early onset Parkinson's disease, exerts a protective effect against DA-induced alpha-synuclein-dependent cell toxicity. Parkin impairs the alpha-synuclein/DAT coupling by interacting with the carboxyl-terminus of the DAT and blocks the alpha-synuclein-induced enhancement in both DAT cell surface expression and DAT-mediated DA uptake. Moreover, we have found that parkin protects against DA-induced cell toxicity in dopaminergic SK-N-SH cells. These findings will help identify the role of these proteins in the etiology and/or maintenance of Parkinson's disease.