We endeavored to determine whether three behavioral effects of melatonin in rodents, i.e., depression of locomotor activity in hamsters, analgesia in mice, and impairment of 3-mercaptopropionic acid (3-MP) convulsions, exhibited the time dependency known to occur for several neuroendocrine effects of the hormone. Activity was monitored and registered by means of an optical actometer, and analgesia was assessed by the hot-plate procedure. Locomotor activity, analgesia, and seizure susceptibility were maximal at the beginning of the scotophase and minimal at noon. The effects of melatonin on the three parameters peaked at early night. The administration of the benzodiazepine antagonist flumazenil, although unable by itself to modify locomotor activity, pain, or seizure threshold, blunted the activity of melatonin. These results suggest that the time-dependent effects of melatonin on specific rodent behaviors may be mediated by central synapses employing gamma-aminobutyric acid (GABA) as an inhibitory transmitter.