The structures capable of synthesizing cyclic GMP in response to nitric oxide in the rat brain were compared relative to the anatomical localization of neuronal nitric oxide synthase. In order to do this, we used brain slices incubated in vitro, where cyclic GMP-synthesis was stimulated using sodium nitroprusside as a nitric oxide-donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Nitric oxide-stimulated cyclic GMP synthesis was found in cells and fibers, but was especially prominent in varicose fibers throughout the rat brain. Fibers containing the nitric oxide-stimulated cyclic GMP production were present in virtually every area of the rat brain although there were large regional variations in the density of the fiber networks. When compared with the localization of nitric oxide synthase, it was observed that although nitric oxide-responsive and the nitric oxide-producing structures were found in similar locations in general this distribution was complementary. Only occasionally was nitric oxide-mediated cyclic GMP synthesis observed in structures which also contained nitric oxide synthase. We conclude that the nitric oxide-responsive soluble guanylyl cyclase and nitric oxide synthase are usually juxtaposed at very short distances in the rat brain. These findings very strongly support the proposed role of nitric oxide as an endogenous activator of the soluble guanylyl cyclase in the central nervous system and convincingly demonstrate the presence of the nitric oxide-cyclic GMP signal transduction pathway in virtually every area of the rat brain.