In HEK 293 cells expressing the human dopamine transporter (DAT), a 10-min incubation with 10 μM cocaine followed by extensive washing resulted in a 30% increase in [³H]dopamine (DA) uptake as well as an increase in cell surface DAT in biotinylation experiments. Consistent with this novel regulation, [³H]DA uptake into synaptosomes prepared from the nucleus accumbens of rats sacrificed 30 min after a single cocaine injection (30 mg/kg) was significantly increased compared to controls (56% increase in V_max, no change in K_m). In addition, DA clearance in the striatum of anesthetized rats was increased after local application of a low (3 pmol) but not high (65 pmol) dose of cocaine, presumably as a result of mobilization of DAT to the cell surface. Cocaine-induced increases in cell surface expression of DAT and associated changes in DA clearance represent a novel mechanism that may play a role in its addictive properties.