Glutamate receptors are known to be densely distributed in the forebrain rewarding circuits, and glutamatergic transmission is actively involved in the regulation of rewarding and reinstating effects of drugs of abuse. Here we investigated the possible involvement of the N-methyl-D-aspartate (NMDA) receptors in the reinstatement of extinguished morphine conditioned place preference (CPP) in rats. We found that previously extinguished morphine (3 mg/kg, i.p.) CPP was markedly reinstated by a priming injection of morphine (2 mg/kg, i.p.) or an acute environmental stressor (forced swim for 10 min), but not by the stress induced by a 24-h food deprivation. Parallel with this, protein levels of the NMDA receptor 2B subunit (NR2B) were elevated in the nucleus accumbens (NAc) and the hippocampus, but not the prefrontal cortex, of reinstated rats. Systemic administration of an NR2B selective antagonist ifenprodil (1, 3, 10 mg/kg, i.p.) attenuated the reinstatement induced by a priming morphine injection, although not by the forced swim. Ifenprodil (2.0 microg/rat) directly injected into the NAc shell or the CA1 region of the dorsal hippocampus produced a similar effect. These results indicate that the NR2B-containing NMDA receptors in the NAc and the dorsal hippocampus play a significant role in mediating the reinstatement of rewarding responses to morphine.