Abstract The present study was designed to provide evidence that the behavioral effects of physostigmine are related to inhibition of brain acetylcholinesterase (AChE). Adult male Holtzman rats were trained to pole jump to a buzzer as the conditioned stimulus and to electric shock to the grid floor as the unconditioned stimulus. All drugs were given s.c. as base. Physostigmine (0.125–0.25 mg/kg) depressed pole jump behavior in methyl atropine (2.1 mg/kg) or saline-treated rats. (−) Hyoscyamine (1 mg/kg) blocked the actions of physostigmine, suggesting a central site of action. Total brain acetylcholine (ACh) and AChE were determined in various groups of animals. The increase in brain ACh and the decrease in AChE activity were negatively correlated in physostigmine-treated rats. These changes were highly correlated during the onset and peak of physostigmine behavioral effects but not during the recovery period. Although (−) hyoscyamine antagonized the behavioral effects of physostigmine, it did not alter the increase in brain ACh or decrease in brain AChE. It is concluded that most of the behavioral effects of physostigmine are related to central AChE inhibition.