Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [ H]NE uptake capacity, an effect characterized by a robust decrease in the V of the transport of [ H]NE. As expected, reserpine did not displace the binding of [ H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [ H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [ H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca /Ca -calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [ H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl-p-tyrosine, increased [ H]NE uptake and eliminated the inhibitory effects of reserpine on [ H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca -independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.