The pineal gland and its hormone melatonin are important in the control of circadian rhythms in birds. Recent research has indicated the presence of high affinity melatonin-binding sites in a wide array of avian cerebral structures. In this study melatonin-binding sites were localized and characterized in the brains of 2-week-old chicks using in vitro autoradiography and image analysis of 2-[125I]iodomelatonin (IMEL) binding. The IMEL binding predominated in the major components of the chick's visual and auditory systems. These brain binding sites showed a daily variation in IMEL binding, with a higher density of binding during the daytime. A significant peak in IMEL binding was observed in the late afternoon, Zeitgeber time 10. This rhythm of IMEL binding continued in constant darkness, with the peak at circadian time 10. The amplitude of the peak was increased in constant darkness in all structures, with the exception of the ectostriatum and neostriatum. Scatchard analysis of the binding revealed an average increase of 45 +/- 5% in the number of binding sites in the daytime vs. nighttime samples, with little change in the binding affinities, indicating that the rhythm in binding is caused by an increase in the total number of binding sites available in the daytime tissue, rather than an increase in the affinity of the binding sites. The data suggest that a rhythm of melatonin sensitivity in a diverse set of cerebral structures may regulate a temporal control of their function.