In the isolated rat middle cerebral artery (MCA) we investigated the role of nitric oxide (NO)/cGMP in the vasodilatory response to extraluminal acidosis. Acidosis increased vessel diameter from 140 ± 27 μm (pH 7.4) to 187 ± 30 μm (pH 7.0, P < 0.01). NO synthase (NOS) inhibition by N ^ω-nitro-l-arginine (l-NNA, 10 μM) reduced baseline diameter (103 ± 20 μm, P < 0.01) and attenuated response to acidosis (9 ± 8 μm). Application of the NO-donors 3-morpholinosydnonimine (1 μM) or S-nitroso- N-acetylpenicillamine (1 μM), or of 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP, 100 μM) reestablished pre-l-NNA diameter at pH 7.4 and reversedl-NNA-induced attenuation of the vessel response to acidosis. Restoration of pre-l-NNA diameter (pH 7.4) by papaverine (20 μM) or nimodipine (30 nM) had no effect on the attenuated response to acidosis. Guanylyl cyclase inhibition with 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (5 μM) or NOS-inhibition with 7-nitroindazole (7-NI, 100 μM) reduced baseline vessel diameter (109 ± 8 or 127 ± 11 μm, respectively) and vasodilation to acidosis, and restoration of baseline diameter with 8-BrcGMP (30 μM) completely restored dilation to pH 7.0. Chronic denervation of NOS-containing perivascular nerves in vivo 14 days before artery isolation significantly reduced pH-dependent reactivity in vitro (diameter increase sham: 48 ± 14 μm, denervated: 14 ± 8 μm), and 8-BrcGMP (30 μM) restored dilation to pH 7.0 (denervated: 49 ± 31 μm). Removal of the endothelium did not change vasodilation to acidosis. We conclude that NO, produced by neuronal NOS of perivascular nerves, is a modulator in the pH-dependent vasoreactivity.