Acetylcholine exerts strong neuromodulating action at cortical and subcortical neurons and networks. Alterations of cortical excitability, induced by acute and repeated administration of the acetylcholinesterase inhibitor rivastigmine were investigated with transcranial magnetic stimulation in healthy volunteers. It was found that rivastigmine had an overall significant effect on the stimulus-response curve with mean values suggesting an enhancement 1.5 and 3 h after a single loading dose of 3 mg rivastigmine and a reduction after 7 days of daily administration of 3 mg rivastigmine. Motor threshold, intracortical inhibition, intracortical facilitation, cortical silent period, M-wave, F-wave and peripheral silent period remained unaffected either by acute or repeated administration of rivastigmine. Our results demonstrate that effects of acetylcholine on motor cortex excitability in healthy subjects are reflected by alterations of the stimulus-response curve. The differential effect between acute and chronic administration of rivastigmine may reflect dynamic properties of different receptor subtypes.