We previously reported that donepezil, a central-type acetylcholinesterase inhibitor, showed neuroprotective action via alpha4-and alpha7-nicotinic acetylcholine receptors against glutamate neurotoxicity in rat cortical culture. The present study was performed to investigate whether the neuroprotective action of acetylcholinesterase inhibitors is accompanied by the alteration of expression and function of nicotinic receptors. Four days treatment with acetylcholinesterase inhibitors (10 microM) enhanced the nicotine-induced increase of the intracellular calcium concentration. Immunoblot analysis revealed that donepezil increased both alpha4 and alpha7 subunit proteins. Donepezil and galanthamine increased the number of cells expressing alpha4- and alpha7-nicotinic receptors in immunocytochemical analysis. We examined whether up-regulation of nicotinic receptors affected the neuroprotective action of acetylcholinesterase inhibitors. Under up-regulating conditions, donepezil and galanthamine exerted neuroprotective action at lower concentrations. These results suggest that donepezil and galanthamine up-regulate nicotinic receptors in cortical neurons, and that the up-regulation of nicotinic receptors may make cortical neurons more sensitive to the neuroprotective action of donepezil and galanthamine.