Melanin concentrating hormone (MCH) binding to central MCH1 receptors leads to an increase in food intake (Hervieu et al., 2000). Although molecular biology techniques have identified MCH containing neurons and mRNA for MCH1 in rat brain (Hervieu et al., 2000), a lack of suitable radioligands and optimised binding conditions have limited the ability to determine MCH1 receptor distribution and compound potencies in native tissues. The aim of this study was to optimise the conditions for [125I]-S36057 binding in sections of rat brain. With local ethical approval, Sprague-Dawley rats (250-350g) were killed by decapitation, brains rapidly removed and snap frozen in isopentane. Cryostat sections (20 µm) of rat brains were pre-incubated in assay buffer containing 25 mM HEPES, 5 mM MgCl2, 0.5% BSA, 1 µM phosphoramidon, 10 µM captopril and 1 mM PMSF, pH 7.4 for 15 min. Subsequently, sections were incubated with 50 pM [125I]-S36057 (Perkin Elmer, UK) for 90 min, in the absence (total binding) or presence (non-specific binding) of MCH (1 µM). Sections were rinsed in 50 mM Tris and apposed to phosphoscreen for 12 hr. For IC50 determination, coronal sections capturing the caudate putamen were incubated with a range of concentrations (10 pM – 10 µM). Specific binding (counts/mm2) was determined using ImageQuant and IC50 analysis was performed using an in-house curve fitting programme. To obtain 65-70% specific binding, greater than the reported 30% in rat brain homogenates (Audinot et al., 2001), it was essential to have 15 min pre-incubation, protease inhibitors and a wash time of 2 x 2 min. There was no difference in specific binding between genders. The highest density of receptors were localised to the caudate putamen, with moderate levels in pre-frontal and frontal cortex, anterior olfactory nucleus, substantia nigra, amygdaloid nuclei, hypothalamic nuclei, brain stem and cerebellar cortex. Low levels of receptors were observed in the thalamus. Receptors were not detected in the deep cerebellar nuclei and inferior colliculus, in contrast to the reported gene expression (Hervieu et al., 2000). Two small molecule MCH1 antagonist, SNAP-94847 and GW-803430 (Rokosz & Hobbs, 2006) inhibited [125I]-S36057 binding with IC50 values ± s.e.mean of 1.26 ± 0.11 nM and 15.1 ± 3.6 nM (all n=4) respectively. In summary, we have reported for the first time, optimised binding conditions using [125I]-S36057 in cryostat sections of rat brain to demonstrate reproducible high specific binding, discrete MCH1 receptor protein localisation and binding affinity of small molecule antagonists. This methodology may prove useful to determine native tissue invitro binding data for lead compounds in other species and tissues.