The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP+) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP+-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP+ produced a concentration- and time-dependent reduction in the uptake of [H-3]dopamine. We observed a significant decrease in [H-3]WIN 35428 binding in the intact cells with MPP+. The saturation analysis of the [H-3]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B-max) with an increase in the dissociation equilibrium constant (K-d) after MPP+ treatment. Furthermore, biotinylation assays confirmed that MPP+ reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP+ may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity. (C) 2003 Elsevier Inc. All rights reserved.