The diurnal variation of [125I]iodomelatonin binding sites in the brain and serum melatonin levels were studied at 4-hour intervals under a 12 h:12 h light:dark cycle in 5-week-old chicks. There was a diurnal rhythm of [125I]iodomelatonin binding capacities in brain membrane preparations with the peak in the light period and the trough in the dark period. However, a diurnal variation with high levels in the dark and low levels in the light was observed in serum melatonin concentrations. The binding capacity of [125I]iodomelatonin was inversely related to the serum melatonin level. Four-week-old chicks were injected with melatonin (1 mg melatonin/kg; i.p.) twice a day (shortly after the light onset and shortly before light offset) for 3 weeks. When chickens were killed at 7 weeks, there was no diurnal rhythm of melatonin in the circulation. In addition, melatonin injection also abolished the diurnal variation of [125I]iodomelatonin binding sites in brain membrane preparations in these animals. This was achieved by decreasing the binding capacity in the light period. Pinealectomy in 2-day-old chickens increased the density of [125I]iodomelatonin binding sites in the brain membrane preparations both at midlight and middark when the chicks were later sacrificed at 6 weeks of age, although the rhythm of binding sites persisted. It is suggested that the [125I]iodomelatonin binding capacity in the chicken brain is regulated by endogenous melatonin and influenced by exogenous melatonin. In addition, the rhythmicity of [125I]iodomelatonin binding sites in the chicken brain is affected by, but not dependent on, pineal melatonin.