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High affinity binding of melatonin in crude membrane preparations of bovine brain was examined by a rapid filtration procedure through Whatman GFB paper. Melatonin binding to medial basal hypothalamic (MBH) membranes attained its maximum at the first, third, and fifth hours of incubation at. 37,18, and 0 C, respectively. Specific binding was linear up to 3 mg membrane protein, was thermolabile, and decreased after incubation with trypsin; it was also pH dependent, the maximum being observed at pH 7.4. Melatonin binding was affected by a variety of ionic manipulations; it was inhibited 55% and 62% after addition of 10 mM KC1 and 120 mM NaCl, respectively, and it was increased 40% and 50% after the addition of 4 or 6 mM CaCl2. Melatonin binding was increased 25% by 1.25 mM MgCl2, whereas it was depressed at higher concentrations. Among the various brain regions studied, melatonin binding was maximal in the MBH; indole binding in occipital and cerebellar cortexes was 73% and 34% that of MBH. Subcellular fractionation studies indicated that about 70% of the binding was located in the 27,000 × g pellet. Scatchard analysis revealed a single population of binding sites with a Kd value of 1.2 ± 0.4 × 10-8 M in three successive experiments; binding site concentration ranged from 8-14 fmol/mg protein. When various indole analogs were tested for their ability to inhibit [3H]-melatonin binding at different concentrations, the following half-maximal inhibition values were obtained: melatonin, 20 nM; 5-methoxytryptophol, 80 nM; 5-methoxyindoleacetic acid, 100 nM; serotonin, 160 nM; 5-hydroxytryptophol, 200 nM; 5-methoxytryptamine, 250 nM; Nacetylserotonin, 250 nM; tryptamine, 250 nM; 2-methyl indole, 1,500 nM; 5-hydroxytryptophan, 1,600 nM; 5-hydroxyindoleacetic acid, 2,000 nM; 6-hydroxymelatonin, >10,000 nM; and indomethacin, > 10,000 nM. These results suggest that melatonin receptors are present in the brain.