The actions of acetylcholine, muscarine and nicotine on the membrane potential of astrocytes of cultured rat brainstem and spinal cord were examined. All 3 cholinergic agonists produced hyperpolarizations of the majority of astrocytes tested, although a small number of cells was depolarized. The hyperpolarizations induced by acetylcholine and muscarine were blocked by the muscarinic antagonist atropine. The nicotinic antagonist mecamylamine antagonized the effects by nicotine in all cells tested. In contrast, mecamylamine completely blocked the acetylcholine induced hyperpolarizations in approximately half of the astrocytes whereas in the remaining cells the response was only reduced. Our electrophysiological studies are consistent with recent autoradiographic binding studies from our laboratory, suggesting the existence of muscarinic and nicotinic receptors on astrocytes.