There is a cholinergic system in large cerebral arteries, but acetylcholine does not mediate the vasodilator response to nerve stimulation. Therefore, the possibility that acetylcholine modulates norepinephrine release must be considered. The effects of cholinergic agonists and antagonists on stimulation-evoked endogenous norepinephrine release from cat and rabbit cerebral arteries were tested at a stimulation frequency of 8 Hz. Acetylcholine (5 X 10(-7) M) decreased norepinephrine release from the cat middle cerebral artery by 40% and by 30% from the rabbit basilar artery. In both species, the effect of acetylcholine was not blocked by hexamethonium but was blocked by atropine. Under the conditions of these experiments, cholinergic and adrenergic nerves are activated simultaneously, so the possibility that cholinergic nerves modulate adrenergic transmission was tested by blocking presynaptic acetylcholine receptors with atropine. However, in both cat and rabbit cerebral arteries, atropine alone had no effect on norepinephrine overflow. These findings suggest that the cholinergic system present in cerebral blood vessels does not modulate norepinephrine release, even though presynaptic cholinergic receptors are present.