This study examined the effects of glutamate receptor agonists on the release of neuropeptide FF-like immunoreactivity from rat spinal dorsal half slices. Glutamate (10 microM) only induced release in Mg(2+)-free medium enriched with glycine (1 microM) and with slight depolarization (15 mM K+). This effect was abolished by the NMDA receptor antagonist, 2-amino-5-phosphonovalerate (100 microM), suggesting major participation of NMDA receptors. The quisqualate and metabotropic receptor agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and trans-1-hydroxy-5-methylisoxazole-4-propionate (t-ACPD) respectively, had no effect at 10 microM. In contrast, NMDA dose dependently stimulated neuropeptide FF release, even in the presence of the Na+ channel blocker, tetrodotoxin (1 microM), suggesting that NMDA receptors involved in the release of neuropeptide FF are mainly located on nerve terminals. The NMDA receptor antagonists, 2-amino-5-phosphonovalerate or (+)-5-methyl-10-11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine (MK-801) (100 microM), blocked the 10 microM NMDA effect. Furthermore, neuropeptide FF-like material inhibited binding of [125I]Y8Fa, a radioiodinated analog of neuropeptide FF, to spinal membranes, suggesting physiological relevance of NMDA-induced release. Taken together, these results suggest a relationship between neuropeptide FF and NMDA receptors in the spinal cord.