The anxiolytic properties of melatonin are revealed by two behavioral studies. In a free exploratory situation, the holeboard test, melatonin decreased head-dip performance. In an unconditioned conflict test, the light/dark box choice situation, melatonin increased the time spent in the lit box as well as the number of transitions between the two compartments. Melatonin was given in a dose range from 0.5 to 5.0 mg/kg body weight i.p. 30 minutes before testing in daytime. Moreover, the anxiolytic activity of diazepam (2.5 mg/kg i.p.) was evaluated and found to be completely inhibited by the specific benzodiazepine antagonist flumazenil (10 mg/kg i.p. 30 minutes before). In the same manner flumazenil counteracted melatonin activity in the two tests. Involvement of the benzodiazepine/GABAergic system in the anxiolytic activity of melatonin is discussed.