The control of arterial vascular tone by acetylcholine contributes to the regulation of cerebral blood flow. We analysed the effects of intraluminal application of acetylcholine (1microM) on the cerebral vascular tone by measuring changes in resistance to perfusion pressure in an isolated guinea-pig brain preparation maintained in vitro by arterial perfusion under constant flow. Acetylcholine induced a reproducible, fast-onset dilation that was prevented by the nitric oxide scavenger Methylene Blue (10microM) and by the muscarinic receptor antagonist atropine (0.1microM). Prolonged arterial perfusion with the nitric oxide synthase inhibitors N-nitro-L-arginine (1mM) and N-nitro-L-arginine methyl ester (30-100microM) induced a slowly developing increase of 25.9+/-13. 44mmHg in vascular tone and blocked the acetylcholine-induced vasodilation. In these experimental conditions, the dilation determined by the nitric oxide donor nitroprusside (0.1microM) was unaffected. In five experiments, the blockade of dilation unmasked a slow acetylcholine-mediated vasoconstriction (14.40+/-3.85mmHg) that was antagonized by atropine.The results demonstrate that acetylcholine exerts two simultaneous and opposite effects on guinea-pig cerebral vessels, characterized by a slow direct constriction concealed in physiological conditions by a fast vasodilation mediated through the release of nitric oxide by endothelial cells. Acetylcholine-mediated increase in vascular tone may play a role in aggravating cerebral perfusion when endothelial cell damage occurs during brain ischemia.