The human dopamine transporter (hDAT) regulates synaptic dopamine (DA) levels and is the site of action of abused and therapeutic drugs. Here we study the effect of a threonine residue (Thr62 in hDAT) that is highly conserved within a canonical phosphorylation site (RETW) in the juxtamembrane N-terminal region of monoamine transporters. In stably transfected human embryonic kidney 293T cells, expression of T62D-hDAT was reduced compared with hDAT or T62A-hDAT. T62D-hDAT displayed dramatically reduced [(3)H]dopamine up-take but exhibited a higher basal dopamine efflux compared with hDAT or T62A-hDAT, as determined by measurements of [(3)H]dopamine efflux and amperometry. The high constitutive efflux in T62D-hDAT precluded the measurement of amphetamine-stimulated [(3)H]dopamine efflux, but when dopamine was added internally into voltage-clamped T62D-hDAT cells, amphetamine-induced efflux comparable with hDAT was detected by amperometry. In accordance with findings that Zn(2+) can rescue reduced DA uptake in mutant transporters that are predominantly inward-facing, micromolar concentrations of Zn(2+) markedly potentiated [(3)H]dopamine uptake in T62D-hDAT and permitted the measurement of amphetamine-stimulated dopamine efflux. These results suggest that T62D-hDAT prefers an inward-facing conformation in the transition between inward- and outward-facing conformations. For T62A-hDAT, however, the measured 50% reduction in both [(3)H]dopamine uptake and [(3)H]dopamine efflux was consistent with a slowed transition between inward- and outward-facing conformations. The mechanism underlying the important functional role of Thr62 in hDAT activity suggested by these findings is examined in a structural context using dynamic simulations of a three-dimensional molecular model of DAT.