A variety of pharmacologic, behavioral, and receptor-binding studies were performed in an effort to determine the mechanism and site of action of nicotine on the rat brain. When nicotine was given into the lateral or fourth ventricles or directly into the lateral vestibular nuclei of rats, it produced a characteristic prostration often accompanied by tonic seizures and body rotation along a longitudinal axis. Of a variety of brain areas studied, the prostration response could only be elicited from the lateral and, to a lesser extent, medial vestibular nuclei. The response could not be produced by a variety of cholinergic agonists or antagonized with nicotinic cholinergic antagonists, with the possible exception of mecamylamine. A good correlation was observed between the ability of nicotine analogues to antagonize the nicotine-induced prostration and their ability to compete with 3H-nicotine binding to rat brain membranes. 3H-nicotine binding had a high affinity, was stereoselective and concentrated in nerve endings and such brain regions as the thalamus, cerebrum, and hippocampus. When nicotine was administered intraventricularly to rats, it significantly elevated the threshold to an aversive shock. It was concluded that many of the central actions of nicotine could not be explained on the basis of traditional nicotinic cholinergic mechanisms.