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Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl2) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl2 and MeHg on [3H]-quinuclidinyl benzilate ([3H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (Bmax) and ligand affinity (Kd). Subsequently, samples were exposed to HgCl2 or MeHg to derive IC50 values and inhibition constants (Ki). Results demonstrate that HgCl2 is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [3H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies.