The molecular basis for substrate translocation in the Na+/Cl--dependent neurotransmitter transporters remains elusive. Here we report novel insight into the translocation mechanism by delineation of an endogenous Zn2+-binding site in the human dopamine transporter (hDAT). In micromolar concentrations, Zn2+ was found to act as a potent, non-competitive blocker of dopamine uptake in COS cells expressing hDAT. In contrast, binding of the cocaine analogue, WIN 35,428, was markedly potentiated by Zn2+. Surprisingly, these effects were not observed in the closely related human norepinephrine transporter (hNET). A single non-conserved histidine residue (His193) in the large second extracellular loop (ECL2) of hDAT was discovered to be responsible for this difference. Thus, Zn2+ modulation could be conveyed to hNET by mutational transfer of only this residue. His375 conserved between hDAT and hNET, present in the fourth extracellular loop (ECL4) at the top of transmembrane segment VII, was identified as a second major coordinate for Zn2+ binding. These data provide evidence for spatial proximity between His193 and His375 in hDAT, representing the first experimentally demonstrated proximity relationship in an Na+/Cl--dependent transporter. Since Zn2+ did not prevent dopamine binding, but inhibited dopamine translocation, our data suggest that by constraining movements of ECL2 and ECL4, Zn2+ can restrict a conformational change critical for the transport process.