In mammals, melatonin receptors MT1 and MT2 are high-affinity G protein-coupled receptors and are thought to be involved in the integration of the melatonin signaling throughout the brain and periphery. In the present study, we describe a new melatonin binding site, named MTx, with a peculiar pharmacological profile. This site had a low affinity for 2-[ 125 I]-melatonin in saturation assays in hypothalamus and retina (pK D = 9.13 ± 0.05 and B max = 1.12 ± 0.11 fmol/mg protein, and pK D = 8.81 ± 0.50 and B max = 7.65 ± 2.64 fmol/mg protein, respectively) and a very high affinity in competition assays for melatonin (pKi = 13.08 ± 0.18) and other endogenous compounds. Using autoradiography, we showed a preferential localization of the MTx in periventricular areas of the sheep brain, with a density 3-8 times higher than those observed for ovine MT 1 . Additionally, using a set of well characterized ligands, we showed that this site did not correspond to any of the following receptors: MT 1 , MT 2 , MT 3 , D 1 , D 2 , noradrenergic, and 5-HT2. Based on its affinity for melatonin, MTx did not seem to be implicated in the integration of cerebral melatonin concentration variations since they were saturating for MTx. Nevertheless, it remained of prime importance because of its periventricular distribution, which was in close contact with the cerebrospinal fluid, and its peculiar pharmacological profile responding to both melatoninergic and serotoninergic compounds. SIGNIFICANCE STATEMENTHerein a putative new melatonin binding site is described in sheep brain parts in close contact with the third ventricle. The characteristics of the pharmacological profile of this site is different from anything previously reported in the literature. The present work forms the basis of future full pharmacological characterization.