alpha-Synuclein (alpha-Syn) regulates catecholaminergic neurotransmission. We demonstrate that alpha-Syn regulates the activity and surface expression of the norepinephrine transporter (NET), depending on its expression levels. In cells co-transfected with NET and low amounts of alpha-Syn, NET activity and cell surface expression were increased and protein interactions with alpha-Syn decreased, compared with cells transfected with NET alone. Converse effects were observed at higher levels of alpha-Syn expression. Treatment with nocodazole and other microtubule (MT) destabilizers abolished the expression-dependent bimodal regulation of NET by alpha-Syn. At low alpha-Syn levels, nocodazole had no effect on NET surface expression or protein interactions, while inducing increases in these measures at higher levels. Cells that were transfected with NET alone displayed no sensitivity to nocodazole, indicating that alpha-Syn expression was necessary for the MT-dependent changes in NET activity. MT destabilizers also caused a significant increase in [(3)H]-NE uptake in brainstem primary neurons and synaptosomes from the frontal cortex, but not striatal synaptosomes. These findings suggest that the surface localization and activity of NET is modulated by alpha-Syn in a manner that is both dependent on interactions with the MT cytoskeleton and varies across brain regions.