1. Acetylcholine introduced into the human cerebral ventricles produced a prolonged and characteristic reaction.2. The responses were a result of the action of acetylcholine on cerebral centres and not of peripheral action after absorption into the blood stream.3. The responses were due to specific actions of acetylcholine; they were prevented or abolished by atropine.4. Eserine injected in small quantities into the ventricles produced a reaction in all respects very closely resembling that to acetylcholine. The site of action was also central.5. Sub‐stimulant quantities of eserine in the ventricles potentiated the action of intraventricular acetylcholine.6. The results of intraventricular eserine suggested that acetylcholine may normally be liberated at some synapses in the brain.7. The nature of the responses to intraventricular acetylcholine has been discussed and particular reference has been made to sleep.Five subjects were patients under charge of Dr. E. Arnold Carmichael and two under charge of Mr. N. M. Dott. We are indebted to them for facilities for this work.