The Adenosine A3 receptor (A3R) and the melanine-concentrating-hormone receptor-1 (MCHR1) are interesting pharmaceutical targets: the A3R is involved in disorders like ischemic events, glaucoma, inflammation and cancer. The MCHR1 plays a role in diseases like depression and obesity.Therefore, potential PET-tracers were developed for the in-vivo visualization of these two receptors, i.e. an A3R antagonist [18F]FE@SUPPY and the MCHR1 antagonists [11C]SNAP-7941 and [18F]FE@SNAP.The objectives of this study were to (1) evaluate different autoradiographic protocols; (2) compare the antagonists with well-known receptor ligands and (3) investigate A3R/MCHR1 expression via autoradiography and Immunohistochemistry (IHC) on human post-mortem brain slices and on coronal and sagittal rat brain slices.The evaluation of different autoradiographic protocols for the A3R showed only slight differences while the autoradiographic protocol for MCHR1 described by Able et al showed no differences to a standard protocol.The comparison of antagonists with other receptor ligands showed partially significant differences between the A3R antagonists FE@SUPPY and MRS1523, which may indicate more off-target binding of MRS1523 in comparison to other AR subtypes.For MCHR1, SNAP-7941 and FE@SNAP showed higher competition potency than the agonist PMC-3886 in coronal and sagittal rat brain. Comparing the two antagonists resulted in opposing results in coronal and sagittal rat brain slices.Both, Autoradiography and IHC indicate amounts of A3R in the analyzed human brain regions in following order (from highest to lowest): putamen, hippocampus, thalamus, cerebellum, nucleus caudatus and cortex.Autoradiography revealed no conclusive order of MCHR1 expression in human brain tissues. IHC showed highest density of the MCHR1 antibody in hypothalamus and thalamus and little density in cerebellum and hippocampus, which is in accordance with literature.