The present study investigated whether the selective nociceptin opioid peptide (NOP) receptor agonist, Ro64-6198, impairs acquisition of fear conditioning through glutamatergic mechanisms. Systemic administration of Ro64-6198 (0.3 and 1mg/kg) or the non-competitive NMDA receptor antagonist, MK-801 (0.03 and 0.1mg/kg) prior to conditioning severely impaired contextual but not cued fear learning in C57BL/6N mice. When administered together at sub-effective doses, Ro64-6198 (0.5mg/kg) and MK-801 (0.05mg/kg), synergistically impaired contextual fear learning, but left cued fear learning intact. We next used the immediate shock deficit paradigm (ISD) to examine the effects of Ro64-6198 and MK-801 on contextual memory formation in the absence of the foot-shock. As expected, naive mice that were shocked briefly after being placed in the training chamber displayed no contextual fear conditioning. This learning deficit was elevated by prior exposure of mice to the training context. Furthermore, administration of Ro64-6198 and MK-801, either separately at amnesic doses (1mg/kg and 0.1mg/kg, respectively) or concomitantly at sub-effective doses (0.5mg/kg and 0.05mg/kg, respectively) significantly reduced the facilitating effects of context preexposure. These findings demonstrate the existence of functional antagonism between NOP and NMDA receptors that predominantly contributes to modulation of conditioned fear learning which involves spatial-processing demands.