Insulin affects brain reward pathways and there is converging evidence that this occurs through insulin regulation of the dopamine (DA) transporter (DAT). In rats made hypoinsulinemic by fasting, synaptosomal DA uptake is reduced. Interestingly, [3H]DA uptake is increased in hypoinsulinemic rats with a history of amphetamine self-administration. The possibility that amphetamine and insulin act in concert to regulate DAT activity prompted this study. Here we show that [3H]DA uptake, measured in vitro and clearance of exogenously applied DA in vivo, is significantly reduced in rats made hypoinsulinemic by a single injection of streptozotocin. Strikingly, amphetamine (1.78 mg/kg, given every other day for 8 days) restored DA clearance in streptozotocin-treated rats but was without effect on DA clearance in saline-treated rats. Basal locomotor activity of streptozotocin-treated rats was lower compared to control rats; however, in streptozotocin-treated rats, hyperlocomotion induced by amphetamine increased over successive amphetamine injections. In saline-treated rats the locomotor stimulant effect of amphetamine remained stable across the four amphetamine injections. These results provide exciting new evidence that actions of amphetamine on DA neurotransmission are insulin-dependent and further suggest that exposure to amphetamine may cause long-lasting changes in DAT function. © 2005 International Society for Neurochemistry.