N-Methyl-D-aspartate (NMDA) receptors regulating the release of [3H]noradrenaline ([3H]NA) and D-[3H]aspartate (D-[3H]Asp) were investigated in superfused slices of rat hippocampus in the presence and absence of nitrergic drugs to examine a possible role for nitric oxide (NO) in the release process. In Mg(2+)-free Krebs-Henseleit buffer, the NMDA-evoked release of [3H]NA and D-[3H]Asp was Ca2+ dependent and inhibited by the NMDA antagonist (+/-)-3-(2-carboxypiperazin-4-yl)propenyl-1-phosphonic acid. NMDA-stimulated release of [3H]NA was tetrodotoxin (TTX; 0.1-2 microM) sensitive, whereas that for D-[3H]Asp was TTX insensitive, indicating that the NMDA receptors involved are differentially localized; those for D-[3H]Asp appear to be presynaptic, whereas those for [3H]NA are extrasynaptic in location. L-Arginine (100 microM), the natural precursor of NO synthesis, enhanced NMDA-evoked release of [3H]NA (100%) and D-[3H]Asp (700%). Exogenous NO donors--sodium nitroprusside, 3-morpholinosyndnomine, and S-nitroso-N-acetylpenicillamine (all 100 microM)--stimulated the NMDA-evoked release. An exception was the inhibition by nitroprusside of NMDA-evoked release of [3H]NA, where the presence of antioxidants may influence channel activity. Inhibitors of NO synthase (NG-nitro-, NG-methyl-, and NG-amino-L-arginine, all 100 microM) attenuated (50-80%) the NMDA-stimulated release of [3H]NA and D-[3H]Asp, as did KN-62 (10 microM), a specific inhibitor of calmodulin kinase II. Our data support roles for the NO transducing system subsequent to the activation of NMDA release-regulating receptors as both an intraneuronal (presynaptically) and an extraneuronal messenger.