Repeated cocaine alters glutamate neurotransmission, in part, by reducing cystine-glutamate exchange via system xc-, which maintains glutamate levels and receptor stimulation in the extrasynaptic compartment. In the present study, we undertook two approaches to determine the significance of plasticity involving system xc-. First, we examined whether the cysteine prodrug N-acetylcysteine attenuates cocaine-primed reinstatement by targeting system xc-. Rats were trained to self-administer cocaine (1 mg/kg/200 microl, i.v.) under extended access conditions (6 h/day). After extinction training, cocaine (10 mg/kg, i.p.) primed reinstatement was assessed in rats pretreated with N-acetylcysteine (0-60 mg/kg, i.p.) in the presence or absence of the system xc- inhibitor (S)-4-carboxyphenylglycine (CPG; 0.5 microM; infused into the nucleus accumbens). N-acetylcysteine attenuated cocaine-primed reinstatement, and this effect was reversed by co-administration of CPG. Secondly, we examined whether reduced system xc- activity is necessary for cocaine-primed reinstatement. To do this, we administered N-acetylcysteine (0 or 90 mg/kg, i.p.) prior to 12 daily self-administration sessions (1 mg/kg/200 microl, i.v.; 6 h/day) since this procedure has previously been shown to prevent reduced activity of system xc-. On the reinstatement test day, we then acutely impaired system xc- in some of the rats by infusing CPG (0.5 microM) into the nucleus accumbens. Rats that had received N-acetylcysteine prior to daily self-administration sessions exhibited diminished cocaine-primed reinstatement; this effect was reversed by infusing the cystine-glutamate exchange inhibitor CPG into the nucleus accumbens. Collectively these data establish system xc- in the nucleus accumbens as a key mechanism contributing to cocaine-primed reinstatement.