Transmural electrical stimulation and nicotine produced a relaxation of dog cerebral artery strips denuded of endothelium, which was abolished by tetrodotoxin and hexamethonium, respectively, and also suppressed by treatment with NG-nitro-L-arginine (L-NA), a nitric oxide (NO) synthesis inhibitor. The inhibition was reversed by L-arginine but not by the D-enantiomer. L-NA also suppressed the endothelium-dependent relaxation by substance P but not the response to NO and nitroglycerin. Treatment with high concentrations of nitroglycerin or sodium nitroprusside markedly inhibited the relaxant response to nicotine, substance P and NO but not the response to papaverine. Slight, slowly developing relaxations caused by L-arginine in the endothelium-denuded arteries were not potentiated by repeated applications of the amino acid or by exposure of the strips for 24 hr to the bathing medium. Ca++ ionophore-induced contractions in the denuded strips were not potentiated by L-NA. Nicotine significantly increased the level of cyclic GMP in the arteries without endothelium; the increment was abolished by treatment with L-NA and hexamethonium. NO does not seem to be synthesized in smooth muscle in an amount sufficient to produce significant relaxation. It may be concluded that NO liberated from vasodilator nerves activates guanylate cyclase in smooth muscle and produces cyclic GMP, resulting in cerebroarterial relaxation.