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Anticholinesterases (antiChEs) are used to treat Alzheimer's disease. The comparative effects of two antiChEs, methanesulfonyl fluoride (MSF) and donepezil, on the extracellular levels of ACh in the hippocampus were investigated by in vivo microdialysis in freely moving rats. MSF at 1 and 2mg/kg produced a dose-dependent increase in ACh efflux from 10min to at least 3hrs after injection. At 2mg/kg, the increase was still present at 24hr. Donepezil at 1mg/kg showed a similar but smaller effect, and, paradoxically, 2mg/kg showed no consistent effect. MSF at 1 and 2mg/kg decreased acetylcholinesterase activity in the hippocampus to 54.8 and 20.1% of control, respectively. These results suggest that MSF is a suitable candidate for the treatment of Alzheimer's disease.