ACUTE incubation of NMDA with neuroblastom a × glioma hybrid (NG108–15) cells or neuroblastoma SK-N-SH cells produced significant attenuation of nociceptin/orphanin FQ (N/OFQ)-induced activation of G protein and inhibition of adenylyl cyclase. The attenuation of N/OFQ signaling by NMDA was dose-dependent, blockable by NMDA antagonists, and not observed in cells lacking NMDA receptors, indicating that the effect of NMDA is mediated by the NMDA receptor. Furthermore, NMDA antagonist pretreatment greatly attenuated N/OFQ-induced acute homologous desensitization of ORL1. Interestingly, the signaling induced by etorphine, an opioid agonist of wide spectrum, was sensitive to NMDA treatment in NG108–15 but insensitive in SK-N-SH cells, suggesting differential modulation of opioid signaling by NMDA. The attenuation effects of NMDA on μ opioid receptor-mediated signaling were also observed.