Melatonin receptors in chick brain were characterized by RRA and localized by in vitro autoradiography, using [125I]melatonin [( 125I]MEL), a biologically active melatonin analog. In membranes from whole brain, radioreceptor studies revealed a high affinity [125I]MEL binding site with an equilibrium dissociation constant of 47.2 +/- 11.5 (mean +/- SEM) pM and a density of 37.8 +/- 8.5 fmol/mg protein. Binding was reversible and competitively inhibited by melatonin and closely related melatonin analogs, but not be norepinephrine or serotonin. In vitro autoradiographic studies of brain revealed a widespread distribution of melatonin receptors. Specific I-MEL binding was observed in retinorecipient and integrative nuclei of the visual system including the avian homolog of the mammalian suprachiasmatic nuclei. Auditory relay nuclei and limbic structures associated with arousal and vocalization were also specifically labeled. This widespread distribution of putative melatonin receptors in chick brain is in marked contrast to the very restricted distribution of melatonin receptors in mammalian brain and suggests that avian sensory systems are affected by melatonin.