One of the major determinants of reinstatement to cocaine use among human addicts is acute reexposure to the drug, which often precipitates cocaine craving and relapse. We used an animal model of cocaine relapse to determine the role of the glutamatergic pathway from the medial prefrontal cortex (mPFC) to the nucleus accumbens in the reinstatement of cocaine-seeking behavior after a cocaine priming injection. Rats were trained to self-administer cocaine intravenously on a second order schedule. Responding was extinguished subsequently by substituting saline for cocaine. During subsequent reinstatement sessions, drug-seeking behavior was assessed after noncontingent priming injections. Results indicated that reinstatement induced by a systemic cocaine injection was blocked by intra-mPFC administration of the dopamine antagonist flupenthixol. Consistent with this finding, administration of cocaine directly into the mPFC reinstated cocaine-seeking behavior. Administration of cocaine into the nucleus accumbens also reinstated drug seeking, whereas microinjection of cocaine into the neostriatum or lateral septum did not. Reinstatement of cocaine seeking induced by intra-mPFC cocaine was blocked by administration of the AMPA receptor antagonist CNQX into the nucleus accumbens. Administration of the NMDA receptor antagonist AP-5 into the nucleus accumbens had variable effects on reinstatement induced by intra-mPFC cocaine in that AP-5 had no effect in some animals but augmented reinstatement in others. Subsequent experiments showed that intra-accumbal microinjection of AP-5 alone dose-dependently reinstated cocaine seeking. These data indicate that the glutamatergic pathway from the mPFC to the nucleus accumbens plays an important role in cocaine priming-induced reinstatement of drug seeking. Moreover, the present results demonstrate that AMPA and NMDA receptors in the nucleus accumbens have opposing roles in the reinstatement of cocaine-seeking behavior.