The neurohormone melatonin is an important signal for both time of day and time of year in many seasonally breeding animals. High densities of melatonin receptors have been found in the suprachiasmatic nucleus, median eminence, and the pituitary gland in almost all mammals investigated so far, and lower densities of melatonin receptors have also been localized to other brain regions varying in a species-specific fashion. Because species-specific differences in receptor distributions have been correlated with differences in behavior and ecology, a comparative study of how melatonin receptors are distributed in vertebrate brains can be useful to the understanding of the functional organization of neural circuits controlling daily and seasonal behaviors. In this study, we localized and characterized melatonin binding sites in the brain of the Mexican free-tailed bat (Tadarida brasiliensis) using in vitro autoradiography with 2-[ 125 I]iodomelatonin. Tadarida brasiliensis is a nocturnal insectivorous mammal that seasonally migrates, reproduces once a year, and exhibits documented sexual dimorphisms in seasonal reproductive behaviors, most notably in courtship vocalizations. Prominent 2-[ 125 I]iodomelatonin binding was found in the median eminence, suprachiasmatic nuclei, and hippocampus, similar to that observed in other mammals. High densities of binding were also localized to structures of the basal ganglia, including the caudate nucleus, putamen, and nucleus accumbens, a feature commonly observed in songbirds but not in mammals. Saturation analysis indicated that the observed binding sites had an affinity for melatonin typical of the binding properties for the Mel 1a receptor subtype. We conclude that melatonin receptor distributions in the Mexican free-tailed bat brain appear to show similarities with the reproductive and circadian systems of other mammals and the basal ganglia of songbirds.