Abstract Functional (CNS) tolerance to the hypnotic effect of barbiturates was determined in vivo by comparing the brain barbiturate level at awakening from a test dose in control rats and rats chronically treated with barbiturate. Approximately two-fold CNS tolerance to barbital was achieved by giving rats increasing concentrations of barbital in their drinking water according to trie schedule of Morgan, Pfeil and Gonzales (1977), but the tolerance was lost after the three days of withdrawal necessary to eliminate the drug from the brain. No significant CNS tolerance to the hypnotic effect of pentobarbital was developed when it was administered in the rats' food or water on various schedules. Administration of pentobarbital by daily injections of the drug in suspension, however, resulted in approximately 1.5-fold CNS tolerance to the hypnotic effect of this barbiturate in 5–10 days. Barbiturates in vitro inhibit K-stimulated ACh release from brain slices; the degree of inhibition by pentobarbital in vitro was not changed after chronic pentobarbital suspension injections in vivo .