One role of the actin cytoskeleton is to maintain the structural morphology and activity of the pre-synaptic terminal. We sought to determine if the actin cytoskeleton plays a role in regulating interactions between the norepinephrine transporter (NET) and alpha-Synuclein (alpha-Syn), two proteins expressed in the pre-synaptic terminal. In cells transfected with either 0.5 microg/mL or 3 microg/mL of alpha-Syn and 1 microg/mL of NET DNA, treatment with cytochalasin D, an actin depolymerizing agent, caused a dose-dependent decrease and increase, respectively, in [3H]-NE uptake. Protein interactions between NET, beta-actin, and alpha-Syn were modified, along with levels of surface transporters. Treatment of primary brainstem neurons and frontal cortex synaptosomes with cytochalasin D caused a 115% and 28% increase, respectively, in NET activity. Depolymerization of both actin and microtubules did not alter NET activity in cells with 0.5 microg/mL alpha-Syn, but caused an increase in [3H]-NE uptake in cells transfected with 3 microg/mL of alpha-Syn and primary neurons. This is the first direct demonstration of NET activity being regulated via actin and modulated by interactions with alpha-Syn.