The characteristics of the binding sites labeled by the radioligand 2-[125I]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2-[125I]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (KD) of 446 ± 55 pMand a total number of binding sites (8max) of 25.4 ± 2.2 fmol/ mg of protein for neuronal retina. For RPE the KD was 34.1 ±2.2 pM and the Bmax 59.5 ± 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2-iodomelatonin < 2-chloromelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin > 6-methoxyharmalan > 5-hydroxytryptamine. Linear regression of log Kivalues from neuronal retina and RPE gave a highly significant dorrelation (r =0.994, n = 8; p <0.001). GTP inhibited specific binding to RPE membranes in a concentration-dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide-binding regulatory protein (G protein), but that in neuronal retina is not.