The primary mechanism for clearance of extracellular dopamine (DA) is uptake mediated by the dopamine transporter (DAT), which is governed, in part, by the number of functional DATs on the cell surface. Previous studies have shown that amphetamine (AMPH) decreases DAT cell surface expression, whereas insulin reverses this effect through the action of phosphatidylinositol 3-kinase (PI3K). Therefore, it is possible that AMPH causes DAT cell surface redistribution by inhibiting basal insulin signaling. Here, we show in a heterologous expression system and in murine striatal synaptosomes that AMPH causes a timedependent decrease in the activity of Akt, a protein kinase immediately downstream of PI3K. This effect was blocked by the DAT inhibitor cocaine, suggesting that AMPH must interact with DAT to inhibit Akt. We also showed that AMPH is able to stimulate Ca2+/calmodulin-dependent kinase II (CaMKII) activity, both in the heterologous expression system as well as in murine striatal synaptosomes. The ability of AMPH to decrease Akt activity was blocked by the CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4- methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93), but not by its inactive analog 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4- chlorocinnamyl)-N-methylbenzylamine (KN92). Furthermore, preincubation with KN93 prevented the AMPH-induced decrease in DAT cell surface expression. Thus, AMPH, but not cocaine, decreases Akt activity through a CaMKII-dependent pathway, thereby providing a novel mechanism by which AMPH regulates insulin signaling and DAT trafficking. Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics.