An ionotropic glutamic acid (Glu) receptor subclass sensitive to N-methyl-D-aspartic acid (NMDA) has three distinct agonist recognition domains for NMDA, glycine (Gly) and polyamines (PAs), while opening of the NMDA channel is inhibited by endogenous cations including Mg²⁺, Zn²⁺ and H⁺. In addition to regulation by these cations, down-regulation is observed following oxidation of sulfhydryl moiety on a redox modulatory site within the NMDA receptor through S-nitrosylation by NO⁺ (nitrosonium ion) donors such as sodium nitroprusside (SNP) and nitroglycerin, which both are clinically used as potent vasodilators. However, SNP, but not S-nitroso-N-acetylpenicillamine and S-nitrose-L-glutathione, markedly inhibits binding of [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine (MK-801) to the open NMDA channel in rat brain synaptic membranes. Moreover, SNP inhibits binding of [³H]Glu and [³H]D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid to the NMDA recognition domain, without affecting binding of [³H]Gly and [³H]5,7-chlorokynurenic acid to the Gly recognition domain. By contrast, pretreatment with SNP leads to a rightward shift of the concentration-response curve for potentiation by Glu of [³H]MK-801 binding, in a manner sensitive to prevention by the disulfide reducing agent, dithiothreitol. Agonists for the PA recognition domain including spermidine and spermine invariably attenuate the potencies of SNP to inhibit binding, furthermore, while putative PA antagonists rather facilitate the inhibition by SNP. These findings suggest that SNP may interfere with opening processes of the native NMDA channel through interactions with both the NMDA and the PA recognition domains, in a manner independent of generation of nitric oxide radicals.