We tested the possibility that endogenous nitric oxide synthase activity regulated NMDA receptors in primary cultured striatal neurons. We monitored NMDA-induced increase in intracellular Ca2+ levels with fura-2 ratio imaging, while nitric oxide synthase activity was either increased with L-arginine (the natural substrate of nitric oxide synthase) or inhibited using nitro-L-arginine (a specific inhibitor of nitric oxide synthase). We found that the NMDA receptor effect was slowly but strongly diminished after an L-arginine (1 mM, 15 min) treatment (L-arginine preincubation reduced the 100 microM NMDA-induced maximal effect by 30-50%). The L-arginine blockade of NMDA receptors was long-lasting but could be partially reversed by hemoglobin (100 microM, 10 min), which binds nitric oxide. This was not observed when the neurons were treated with L-arginine together with nitro-L-arginine. Our data strongly suggest that physiological nitric oxide synthase activity could regulate NMDA receptors.