We have localized and characterized 2–125I-iodomelatonin binding sites in the chicken brain using in vitro quantitative autoradiography. Binding sites were widely distributed throughout the chicken brain, predominantly in regions associated with the visual system. The specific binding of 2–125I-iodomelatonin to discrete chicken brain areas was found to be saturable, reversible, and of high affinity. The specific binding of 2–125I-iodomelatonin (75 pm) was quantitated for 40 identifiable brain regions. Eight brain regions were chosen for binding characterization and pharmacological analysis: optic tectum, Edinger- Westphal nucleus, oculomotor nucleus, nucleus rotundus, ventral supraoptic decussation, ventrolateral geniculate nucleus, neostriatum, and ectostriatum. These regions showed no rostral-caudal gradient in 2- 125I-iodomelatonin specific binding, and saturation analysis revealed a single class of high-affinity sites with KD values in the range of 33- 48 pM and receptor site density (Bmax) ranging from 31 to 58 fmol/mg protein. Competition experiments carried out with various indoles revealed a similar order of pharmacological affinities in these areas: melatonin greater than 6-chloromelatonin greater than methoxyluzindole greater than N-acetylserotonin greater than luzindole much greater than 5-HT greater than 5-methoxytryptamine. The affinity constants determined by quantitative autoradiography for these compounds to compete for 2–125I-iodomelatonin binding in the optic tectum correlated well with the affinities in chicken brain membranes at 25 degrees C (r = 0.966; slope = 0.845; n = 7) and 0 degree C (r = 0.946; slope = 0.379; n = 7), chicken retinal membranes (r = 0.973; slope = 0.759; n = 7), and the potency or affinity of these compounds to affect the calcium-dependent release of 3H-dopamine from the rabbit retina (r = 0.902; slope = 0.506; n = 6). We conclude that the high-affinity sites labeled by 2–125I-iodomelatonin in various chicken brain areas have identical binding and pharmacological characteristics to the ML-1 melatonin binding site previously described in chicken brain and retinal membranes and to the ML-1 melatonin receptor modulating dopamine release from the retina. In the chicken brain, the ML-1 receptor site may mediate functional responses regulated by melatonin.