Interactions between the serotonergic and cholinergic systems are known to occur and are believed to play a role in the mechanism underlying both major depression and Alzheimer's disease. On a molecular level, studies suggest that acetylcholine (ACh) increases serotonin (5-HT) release through nicotinic receptors located at nerve terminals. The aim of the present study was to determine in which areas and to what extent 5-HT mediates the neuronal response to ACh release. For this purpose, neuronal activity was measured in rats with rivastigmine-induced elevated ACh levels after a 95% 5-HT depletion obtained by dosing p-chlorophenylalanine followed by D,L-fenfluramine. Neuronal activation was quantified by stereological measurements of c-Fos immunoreactivity. The brain areas examined were medial prefrontal cortex, septum, dorsal hippocampus, and dorsal raphe nucleus. Rivastigmine significantly increased c-Fos immunoreactivity in medial prefrontal cortex and the hippocampus, but not in the septum and dorsal raphe nucleus. 5-HT depletion decreased ACh-induced c-Fos immunoreactivity in the dentate gyrus. By contrast, 5-HT depletion had no effect on the ACh-induced activity in the other brain areas examined. It is concluded that 5-HT mediates part of the ACh-induced hippocampal neuronal activation, possibly mediated via locally released 5-HT.