Donepezil, an acetylcholinesterase (AChE) inhibitor used for treating Alzheimer's disease patients, is thought to act by increasing brain extracellular acetylcholine (ACh), and ACh binding to cholinergic receptors. Muscarinic receptors are coupled to cytosolic phospholipase A2 (cPLA2) activation and arachidonic acid (AA) release from synaptic membrane phospholipid. This activation can be imaged in rodents as an AA incorporation coefficient k*, using quantitative autoradiography. Acute and chronic effects of donepezil on the AA signal, k* for AA, were measured in 81 brain regions of unanesthetized rats. Twenty min after a single oral dose (3.0 mg/kg) of donepezil, k* was increased significantly in 37 brain regions, whereas k* did not differ from control 7 h afterwards or following chronic (21 days) of donepezil. Pretreatment with atropine prevented the 20-min increments in k* following donepezil. Donepezil also increased the brain ACh concentration and reduced brain AChE activity, but did not change cPLA2 activity, regardless of administration regimen. These results show that donepezil acutely increases the brain AA signal that is mediated by ACh acting at muscarinic receptors, but that this signal is rapidly desensitized despite continued elevated brain ACh concentration. In contrast, the AA signal in response to arecoline was not altered following donepezil.