The characteristics of the binding sites for 2-[125I]iodomelatoion in were studied in chicken brain membranes during development. Specific binding, defined using cold melatonin (1 μM), was detected as early as 8-day-old embryos. Scatchard analysis of saturation experiments showed that 2-[125I]iodomelatonin binds to a single class of site at all ages tested (8-day-old embryos to 3-month-old chicks). Binding affinity (Kd) did not change during development (18–31 pM), but the maximal number of binding sites (Bmax) increased until embryonic day 18, and then remained relatively constant until 30 days of age. A further increase in Bmax was seen at 3 months of age. Guanosine 5′-triphosphate (GTP, 1 mM) inhibited 2-[125I]iodomelatonin binding at all ages suggesting that the melatonin binding site is in coupled to a guanine nucleotide binding protein at a very early stage of development. Competition experiments with a number of melatonin analogues indicated that the binding site detected in the brain at embryonic day 8 was pharmacologically identical to that observed 15 days after hatching.