Opioid receptor agonists and antagonists have profound effects on cocaine-induced hyperactivity and conditioned reward. Recently, the role specifically of the mu opioid receptor has been demonstrated based on the finding that i.c.v. administration of the selective mu opioid receptor antagonist, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), can attenuate cocaine-induced behaviors. The purpose of the present study was to determine the location of mu opioid receptors that are critical for cocaine-induced reward and hyperactivity. Adult male Sprague-Dawley rats received injections of CTAP into the caudate putamen, the rostral or caudal ventral tegmental area (VTA) or the medial shell or core of the nucleus accumbens prior to cocaine to determine the role of mu opioid receptors in cocaine-induced reward and hyperactivity. Cocaine-induced reward was assessed using an unbiased conditioned place preference procedure. Results demonstrate that animals pre-treated with CTAP into the nucleus accumbens core or rostral VTA, but not the caudal VTA, caudate putamen or medial nucleus accumbens shell, during conditioning with cocaine showed an attenuation of the development of cocaine-induced place preference. In contrast, CTAP injected into the nucleus accumbens shell but not the core attenuated the expression of cocaine place preference. Intra-nucleus accumbens core, caudate putamen or caudal VTA CTAP significantly attenuated cocaine-induced hyperactivity. In addition, the number of cFos positive cells was increased in the motor cortex, medial and ventromedial aspects of the nucleus accumbens shell, basolateral amygdala and caudal VTA during the expression of cocaine place preference, and this increase was attenuated in the animals that received intra-accumbens core CTAP during daily cocaine conditioning. These results demonstrate the importance of mu opioid receptors in the nucleus accumbens and VTA in cocaine-induced reward and hyperactivity and suggest that some aspects of the behavioral effects of cocaine are mediated by endogenous activation of mu opioid receptors in these brain regions.