Supraspinal opioid analgesia is mediated in part by connections between the periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Morphine analgesia elicited from the PAG is respectively decreased by selective serotonergic and opioid receptor antagonists administered into the RVM, and increased by RVM neurotensin antagonists. Since glutamate and excitatory amino acid (EAA) receptors are also active in the RVM, the present study evaluated whether either competitive (AP7) or non-competitive (MK-801) N-methyl-D-aspartate (NMDA) antagonists or a kainate/AMPA (CNQX) antagonist microinjected into the RVM altered morphine (2.5 micrograms) analgesia elicited from the PAG as measured by the tail-flick and jump tests. Mesencephalic morphine analgesia was markedly reduced on both tests after RVM pretreatment with either AP7 (0.01-1 microgram, 0.08-7.8 nmol) or MK-801 (0.03-3 micrograms, 0.04-4.4 nmol). In contrast, small but significant reductions in mesencephalic morphine analgesia occurred on the jump test following CNQX (0.5 microgram, 2.2 nmol) in the RVM. NMDA antagonists did not markedly alter either basal nociceptive thresholds following RVM administration, or mesencephalic morphine analgesia following administration into medullary placements lateral or dorsal to the RVM. These data implicate EAA and particularly NMDA receptors in the RVM in modulating the transmission of opioid pain-inhibitory signals from the PAG.