The use of acetylcholinesterase (AChE) inhibitors is the primary therapeutic strategy in the treatment of Alzheimer's disease. However, these drugs have been reported to have effects beyond the simple stimulation of neuronal acetylcholine receptors (AChRs) by elevated acetylcholine (ACh), interfering directly with the nAChR. Therefore, a pure pharmacological blockade of AChE is not usually obtained. In this study, the patch-clamp technique was utilized to determine the effects of methamidophos, a pesticide that is considered a selective AChE inhibitor, on nAChRs of substantia nigra dopaminergic neurons. In spite of the fact that methamidophos has been reported to be devoid of direct nicotinic actions, our main observation was that it selectively and reversibly blocked nAChR responses, without directly affecting the holding current. Methamidophos produced a downward shift in the dose response curve for nicotine; the mechanism accounting for this non-competitive antagonism was open channel block, in view of its voltage dependence. Pre-treatment with vesamicol did not prevent the reduction of nicotine-induced currents, indicating that the effect on nAChRs was independent from the activity of methamidophos as a cholinesterase inhibitor. Our results conclude that methamidophos has a complex blocking action on neuronal nAChRs that is unlinked to the inhibition of AChE. Therefore, it should not be considered a selective AChE inhibitor and part of its toxic effects could reside in an interference with the nicotinic neurotransmission.