The present report describes the cellular and subcellular distribution pattern of immunoreactivity to M35, a monoclonal antibody raised against purified muscarinic acetylcholine receptor protein, in astrocytes in the cerebral cortex of young and aged rats. Most M35-positive astrocytes were localized in the superficial layers of the cortex and part of the corpus callosum. At the ultrastructural level, immunoprecipitates were localized in the Golgi complexes, but the nucleus, rough endoplasmic reticulum, mitochondria, and microfilaments were generally free of labeling. Labeling was also present associated to the cell membrane, although without the characteristic immunoreactive postsynaptic membrane thickening found in neuronal structures. In aging rats of 30-34 months, the number of M35-labeled astrocytes doubled, whereas the neuronal staining slightly decreased in the same region in half of the animals studied. Fluorescent double-labeling for M35 and GFAP, an astrocytic microfilament protein, revealed that all M35-positive glial cells express GFAP and, conversely, that almost all GFAP glial cells were M35-immunostained. Based on the high incidence of coexpression of mAChRs and GFAP, both proteins may be functionally linked to each other. Rough semiquantitative estimates revealed that in young adult rats the GFAP/M35-immunoreactive astrocytes made up approximately one fifth of all cortical astrocytes. An important aspect of the presently demonstrated immunoreactivity of astroglia to mAChR proteins is its labeling in situ instead of in tissue culture. This finding may further support investigation, e.g., on anatomical relations of astroglia with neuronal and vascular elements, and its reactivity in experimental conditions.