AbstractCocaine binds to the dopamine (DA) transporter (DAT) to regulate cocaine reward and seeking behavior. Zinc (Zn2+) also binds to the DAT, but the in vivo relevance of this interaction is unknown. We found that Zn2+concentrations in postmortem brain (caudate) tissue from humans who died of cocaine overdose were significantly lower than in control subjects. Moreover, the level of striatal Zn2+content in these subjects negatively correlated with plasma levels of benzoylecgonine, a cocaine metabolite indicative of recent use. In mice, repeated cocaine exposure increased synaptic Zn2+concentrations in the caudate putamen (CPu) and nucleus accumbens (NAc). Cocaine-induced increases in Zn2+were dependent on the Zn2+transporter 3 (ZnT3), a neuronal Zn2+transporter localized to synaptic vesicle membranes, as ZnT3 knockout (KO) mice were insensitive to cocaine-induced increases in striatal Zn2+. ZnT3 KO mice showed significantly lower electrically evoked DA release and greater DA clearance when exposed to cocaine compared to controls. ZnT3 KO mice also displayed significant reductions in cocaine locomotor sensitization, conditioned place preference (CPP), self-administration, and reinstatement compared to control mice and were insensitive to cocaine-induced increases in striatal DAT binding. Finally, dietary Zn2+deficiency in mice resulted in decreased striatal Zn2+content, cocaine locomotor sensitization, CPP, and striatal DAT binding. These results indicate that cocaine increases synaptic Zn2+release and turnover/metabolism in the striatum, and that synaptically released Zn2+potentiates the effects of cocaine on striatal DA neurotransmission and behavior and is required for cocaine-primed reinstatement. In sum, these findings reveal new insights into cocaine’s pharmacological mechanism of action and suggest that Zn2+may serve as an environmentally derived regulator of DA neurotransmission, cocaine pharmacodynamics, and vulnerability to cocaine use disorders.