The relaxant effect of hypercapnia (15% CO2) was studied in isolated circular segments of rat basilar arteries with intact endothelium. The nitric oxide synthase inhibitor nitro-l-arginine (l-NOARG) and the cytosolic guanylate cyclase inhibitor methylene blue (MB), significantly reduced this relaxation by 54% and 70%, respectively. The effect of l-NOARG was completely reversed by l-arginine. Blockade of nerve excitation with tetrodotoxin (TTX) had no affect on the 15% CO2 elicited vasodilatation. Measurements of cGMP in vessel segments showed no significant increase in cGMP content in response to hypercapnia. l-NOARG and MB, but not TTX, significantly reduced the basal cGMP content in cerebral vessels. Adding 1.5% halothane to the incubation medium did not result in a significant increase in cGMP content. Lowering the pH by cumulative application of 0.12 m HCl resulted in relaxation identical to that obtained by lowering the pH with 15% CO2. In vessel segments in which the endothelium had been removed beforehand 15% CO2 induced relaxation that was not different from that seen in vessels with intact endothelium. l-NOARG had no affect in endothelium denuded vessels. The results suggest that high CO2 elicits vasodilatation of isolated rat basilar arteries by a mechanism independent of nitric oxide synthase (NOS) activity. The markedly reduced basal cGMP levels in cerebral vessels by l-NOARG and MB suggest that there exists a basal NO formation in the cerebral vessel wall.