An unbiased place preference conditioning procedure was used to examine the neural substrates mediating the aversive effects of opioid antagonists in the rat. Microinjection of the non-selective opioid receptor antagonist naloxone into either the ventral tegmental area (VTA) or the nucleus accumbens (NAc) produced significant aversions for an environment previously paired with its administration. The minimum doses producing these effects were 10.0 and 7.5µg, respectively. Microinjections into either the caudate/putamen or medial prefrontal cortex were without effect. Place aversions of equivalent magnitude were also observed in response to the intra-VTA or intra-NAc administration of the highly selective mu opioid receptor antagonist CTOP. Doses as low as 0.3µg resulted in significant effects. 6-hydroxydopamine lesions of the NAc attenuated the aversive effect of intra-VTA CTOP. Such lesions did not modify the aversive effects of intra-NAc CTOP; they also failed to modify the aversive effects of systemically administered naloxone. These data demonstrate that the blockade of either VTA or NAc mu opioid receptors is sufficient for the expression of the aversive effects of opioid antagonists. Furthermore, they suggest that whereas the aversive effects of intracranially applied opioid antagonists may involve both a mesolimbic DA-dependent (VTA) and independent (NAc) component, the aversive effects produced by systemically administered opioid receptor antagonists are independent of mesolimbic DA neurons.