In the guinea pig basilar artery, acetylcholine and the calcium ionophore A23187 induced endothelium-dependent relaxations, which were not significantly affected by the nitric oxide (NO) synthase inhibitor N<sup>ω</sup>-nitro- L-arginine ( L-NOARG; 0.3 m M) or the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one; 1-10 µ M), or by these inhibitors combined. However, acetylcholine (10 µ M) and A23187 (3 µ M) each significantly increased the tissue level of cGMP in the absence but not in the presence of L-NOARG, suggesting that NO is released from the vascular endothelium in this blood vessel. Treatment with the potassium (K) channel inhibitors charybdotoxin (0.1 µ M) plus apamin (0.1 µ M), a toxin mixture previously shown to inhibit relaxations mediated by endothelium-derived hyperpolarizing factor (EDHF) in this artery, had no effect on the A23187-induced relaxation but slightly inhibited the response to acetylcholine (E<sub>max</sub> was reduced by 24%). When the action of EDHF was prevented by these K channel inhibitors, the remaining relaxation was abolished by either ODQ (1 µ M) or L-NOARG (0.3 m M), indicating that NO, apart from EDHF, contributes to the endothelium-dependent relaxations. Furthermore, ODQ (10 µ M) abolished the relaxation induced by the NO donor S-nitroso-N-acetylpenicillamine. Thus, activation of soluble guanylate cyclase seems to be the only mechanism through which NO causes relaxation in this artery. When vessels were exposed to grave hypoxia (pO<sub>2</sub> = 6 mm Hg), the NO-mediated relaxation (induced by acetylcholine in the presence of charybdotoxin plus apamin) disappeared. In contrast, EDHF-mediated responses (elicited by acetylcholine in the presence of L-NOARG) were only marginally affected by hypoxia (E<sub>max</sub> was reduced by 16%). 17-Octadecynoic acid (50 µ M) and 5,8,11,14-eicosatetraynoic acid (10 µ M), inhibitors of cytochrome P<sub>450</sub>-dependent oxidation of arachidonic acid, failed to inhibit the acetylcholine-induced relaxation in the presence of L-NOARG. The cytochrome P<sub>450</sub>-dependent arachidonic acid metabolite 11,12-epoxyecosatrienoic acid (0.3–3.0 µ M) had no relaxant effect per se. In conclusion, EDHF and NO are both mediators of endothelium-dependent relaxations in the guinea pig basilar artery. However, during grave hypoxia, EDHF alone mediates acetylcholine-induced relaxation. The results further suggest that EDHF is not a metabolite of arachidonic acid formed by cytochrome P<sub>450</sub> mono-oxygenase or generated by another oxygen-dependent enzyme in this artery.