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-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 reversed L-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.