Cerebral blood flow (CBF) was estimated from measurements of internal carotid blood flow and sagittal sinus blood flow in mechanically ventilated rabbits under 70% N2O-30% O2. Intravenously administered physostigmine, a cholinesterase inhibitor, increased CBF under normocapnia and enhanced the cerebral vasodilatation of hypercapnia, but did not alter the cerebral metabolic rate of oxygen (CMRO2). The cerebrovascular effects of physostigmine were antagonized by atropine but not by dihydro-beta-erythroidine, a nicotinic blocker. Neostigmine, a quaternary cholinesterase inhibitor that does not cross the blood-brain barrier, showed no cerebrovascular effects. It is concluded that the cholinergic cerebral vasodilatation does not depend on cerebral metabolic activation, and that the cholinergic receptors involved are muscarinic and located beyond the blood-brain barrier.