The roles that the pineal gland and its hormone melatonin play in the regulation of circadian rhythmicity and photoperiodism vary among vertebrate species. Recently, putative sites of melatonin action have been elucidated in several avian and mammalian species by application of in vitro binding of a radioiodinated melatonin agonist, 2[125I]iodomelatonin (IMEL) and autoradioradiography. These studies in mammals, birds and reptiles have indicated profound differences in the distribution of IMEL binding between these diverse groups, suggesting that these large differences in binding may reflect differences in melatonin function. The present study was performed to determine systematically whether the variance in IMEL binding among avian species corresponds to changes in circadian organization and/or phylogenetic relationships. The distribution of specific IMEL binding was determined in the brains from birds belonging to 14 different species in 5 Orders (Psittaciformes, Passeriformes, Columbiformes, Galliformes and Anseriformes) using in vitro binding, autoradiography and computer-assisted image analysis. The distribution was compared to a similar study in 3 species of turtles as an outgroup. The data indicated IMEL binding in retinorecipient structures of the circadian, tectofugal, thalamofugal and accessory optic visual pathways in all avian species. Relay nuclei and integrative structures of the tectofugal, thalamofugal, accessory optic, and limbic systems, however, bound the hormone to varying degrees. In turtles, binding was observed in retinorecipient structures of the thalamofugal visual pathway and in retinorecipient and integrative areas of the tectofugal visual pathway. No binding was observed in the pineal gland, tuberal hypothalamus or adenohypophysis in any avian or testudine species. This distribution is drastically different from that observed in mammals, where binding predominates in the pars tuberalis of the adenohypophysis and in the suprachiasmatic nucleus, suggesting that the circadian system may influence a wide array of sensory and integrative functions in birds and reptiles through the circadian secretion of melatonin, but that this capacity has been lost in mammals.