In an investigation aimed at comprehensive mapping of the adult human brain with respect to receptor sites for the pineal hormone melatonin, we consistently observed specific binding in the cerebellum. Autoradiography and in vitro binding analysis with 125I-labeled melatonin were used to examine the location and the properties of these binding sites. In all cerebellar lobes, highest-density specific binding was localized to the external zone of the molecular layer. The binding was rapid, saturable, displaceable, specific and of high affinity. Physiological concentrations of NaCl decreased the affinity, while presence of calcium ions promoted it. The non-hydrolyzable GTP analog, GTP gamma S, inhibited binding in a dose-dependent manner and provoked a shift towards low affinity. The results strongly suggest that these binding sites may be functional melatonin receptors, and indicate that the adult human cerebellum is a target of melatonin, the pineal hormone involved in the control of the circadian timing.