High-affinity binding of melatonin to crude membrane preparations of bovine pineal gland was examined by a rapid filtration procedure through Whatman GFB paper. Maximal melatonin binding was attained in 60 min at 37 °C, in 2 h at 25 °C and in 5 h at 0 °C; at 25 and 37 °C it was 36 and 42% of that at 0 °C. Specific binding was thermolabile and decreased following incubation with trypsin; it was also pH dependent, the maximum being observed at physiological pH. Melatonin binding was inhibited by the addition of monovalent or divalent ions to the incubation buffer. Subcellular fractionation studies indicated that 39 and 50% of binding was located in the pellets at 900 and 27,000 g whereas 11% was detected in the microsomal pellet. Scatchard analysis revealed a single population of binding sites with K<sub>d</sub> = (7.0 ± 1.5) 10<sup>-7</sup> M (mean ± SEM, n = 4); binding site concentration ranged from 185 to 356 fmol/mg of protein. When various melatonin analogues were tested for their ability to inhibit (<sup>3</sup>H)-melatonin binding, the following Ki values (10<sup>-7</sup>M), were obtained: N-acetyl-serotonin 120, serotonin 130, 2-methyl indole 154, 5-hydroxytryptophol 218, 5-methoxytryptamine 266, 5-methoxytryptophol 660, tryptamine 1,740, 5-hydroxyindole acetic acid 3,455, 5-methoxyindole acetic acid 12,690, 5-hydroxytryptophan 13,600, and 6-hydroxymelatonin 55,550. These results suggest that melatonin receptors may be present in the pineal gland.