The critical observation in the pathology of Parkinson's disease (PD) is that neurodegeneration is largely restricted to dopaminergic neurons that develop cytoplasmic inclusions called Lewy bodies. These aggregations contain the protein α-synuclein. Furthermore, it is becoming apparent that α-synuclein expression levels are a major factor in PD pathogenesis. Patients with additional copies of the α-synuclein gene develop PD with a severity proportional to levels of α-synuclein overexpression. Similarly, overexpression of α-synuclein in in vitro and in vivo models has been shown to be toxic. However, little is known about the effects of reducing α-synuclein expression in human neurons. To investigate this, we have developed a system in which levels of α-synuclein can be acutely suppressed by using RNA interference (RNAi) in a physiologically relevant human dopaminergic cellular model. By using small interfering RNA (siRNA) molecules targeted to endogenous α-synuclein, we achieved 80% protein knockdown. We show that α-synuclein knockdown has no effect on cellular survival either under normal growth conditions over 5 days or in the presence of the mitochondrial inhibitor rotenone. Knockdown does, however, confer resistance to the dopamine transporter (DAT)-dependent neurotoxin N-methyl-4-phenylpyridinium (MPP+). We then demonstrate for the first time that α-synuclein suppression decreases dopamine transport in human cells, reducing the maximal uptake velocity (Vmax) of dopamine and the surface density of its transporter by up to 50%. These results show that RNAi-mediated α-synuclein knockdown alters cellular dopamine homeostasis in human cells and may suggest a mechanism for the increased survival in the presence of MPP+, a toxin used extensively to model Parkinson's disease. © 2006 Wiley-Liss, Inc.