Melatonin receptor sites were characterized in the brains of four avian species (duck, goose, pigeon, and turkey) by anin vitrobinding technique, using 2- [ 125 I]iodomelatonin as a labeled ligand. The specific binding of 2-[ 125 I]iodomelatonin to avian brain membranes was found to be stable, saturable, reversible, and of high affinity. Scatchard analysis revealed that 2-[ 125 I]iodomelatonin binds to a single class of sites in brain membrane preparations of these species with high affinity (affinity constantsK d between 153 and 289 pM) and low capacity (total number of binding sitesB max between 5.07 and 10.15 fmol/mg protein). Competition studies showed the following relative order of potency of indolic compounds to inhibit the specific 2-[ 125 I]iodomelatonin binding: melatonin >>N-acetyltryptamine >N-acetyl-5-hydroxytryptamine > 5-hydroxytryptamine >>> 5-methoxytryptamine. We conclude that the 2-[ 125 I]iodomelatonin-binding sites in brains of duck, goose, pigeon, and turkey have similar binding and pharmacological characteristics, and may represent ML-1 melatonin receptor sites.