The dopamine (DA) transporter (DAT) mediates the removal of released DA. DAT is the major molecular target responsible for the rewarding properties and abuse potential of the psychostimulant amphetamine (AMPH). AMPH has been shown to reduce the number of DATs at the cell surface, and this AMPH-induced cell surface DAT redistribution may result in long-lasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wildtype human DAT (WT-hDAT). However, AMPH did not induce cytosolic redistribution in an uptake-impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn2+) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn2+ and AMPH consistently reduced WT-hDAT trafficking but stimulated cytosolic redistribution of Y335A-hDAT. Furthermore, direct intracellular application of AMPH, via a whole-cell patch pipette, stimulated the trafficking of Y335A-hDAT. Taken together, these data suggest that the DAT transport cycle is not required for AMPH-induced down-regulation and that an increase of intracellular AMPH is an essential component of DAT redistribution. Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics.