The dopamine transporter (DAT), a membrane protein specifically expressed by dopaminergic neurons and mediating the action of psychostimulants and dopaminergic neurotoxins, is regulated by Zn(2+) which directly interacts with the protein. Herein, we report a host-cell-specific direction of the Zn(2+) effect on wild type DAT. Whereas low mumolar Zn(2+) decreased dopamine uptake by DAT expressing HEK293 cells, it stimulated uptake by DAT expressing SK-N-MC cells. Inhibition or stimulation was lost in a DAT construct without the binding site for Zn(2+). Also reverse transport was differentially affected by Zn(2+), dependent on whether the DAT was expressed in HEK293 or SK-N-MC cells. Pre-treatment of DAT expressing cells with phorbol-12-myristate-13-acetate, an activator of protein kinase C, attenuated the inhibitory effect of Zn(2+) on uptake in HEK293 cells and increased the stimulatory effect in SK-N-MC cells. Patch-clamp experiments under non-voltage-clamped conditions revealed a significantly higher membrane potential of HEK293 than SK-N-MC cells and a reduced membrane potential after phorbol ester treatment. Lowering chloride in the uptake buffer switched the stimulatory effect of Zn(2+) in SK-N-MC cells to an inhibitory, whereas high potassium depolarization of HEK293 cells switched the inhibitory effect of Zn(2+) to a stimulatory one. This study represents the first evidence that DAT regulation by Zn(2+) is profoundly modulated by the membrane potential and chloride.