Although it is clear that amphetamine-induced dopamine (DA) release mediated by the dopamine transporter (DAT) is integral to the behavioral actions of this psychostimulant, the mechanism of this release is not clear. In this study, we explored the requirement for intracellular Ca2+ in amphetamine-induced DA efflux and currents mediated by the human DAT. The patch-clamp technique in the whole-cell configuration was used on Na+ and DA-preloaded human embryonic kidney 293 cells stably transfected with the human DAT (hDAT cells). Chelation of intracellular Ca2+ by inclusion of 50 μM BAPTA in the whole-cell pipette reduced the voltage-dependent amphetamine-induced hDAT current, with the greatest effect seen at positive voltages. Likewise, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′- tetraacetic acid (BAPTA) reduced amphetamine-induced DA efflux as measured by amperometry. Furthermore, preincubation of the cells with 50 μM BAPTA acetoxy methyl ester (AM) or thapsigargin also blocked amphetamine-induced release of preloaded N-methyl-4-[3H]phenylpyridinium from superfused hDAT cells. BAPTA-AM also reduced DA release from striatal synaptosomes. Amphetamine also led to an increase in intracellular Ca2+ that was blocked by prior treatment with 5 μM thapsigargin or 10 μM cocaine. These studies demonstrate that amphetamine-induced DAT-mediated currents and substrate efflux require internal Ca2+ and that amphetamine can stimulate dopamine efflux by regulating cytoplasmic Ca2+ levels through its interaction with DAT.