Activation of N-methyl-D-aspartate (NMDA) receptors in rat cerebellum leads to the release of endothelium-derived relaxing factor, now identified as nitric oxide (NO), a stimulator of soluble guanylate cyclase. L-NG-monomethylarginine (L-NMMA), which blocks NO synthesis from L-arginine in several tissues, including a crude synaptosomal preparation from brain, inhibited the elevation of cyclic GMP induced by NMDA in rat cerebellar slices. D-NMMA was ineffective. L-Arginine, but not its D enantiomer, augmented the response to NMDA and reversed the inhibition by L-NMMA. The results indicate that stimulation of NMDA receptors results in the activation of the enzyme which catalyzes the formation of NO from L-arginine.