Using combined double immunofluorescence and laser confocal microscopy, we studied the common cellular localization of cholinergic muscarinic receptors (mAChRs) and aquaporin-4 water channels (AQP4) in the cortex, the corpus callosum and in ependymal cells of the rat brain. In the cortex, AQP4 staining was restricted to the perivascular end-feet of astrocytes. It was more widely distributed on the astrocytes of the corpus callosum. On astrocytes, mAChRs were often present in regions immunoreactive to AQP4. Ependymal cells bordering the third ventricle were also stained by both antibodies. The double staining of mAChRs with AQP4 on two different cell-types might indicate that further interactions exist which may be important in the regulation of water and electrolyte movements in the brain.