This study describes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) in the medial septum (MS), employing the monoclonal antibody M35 raised against purified mAChR-protein. mAChR-positive neurons are found throughout the MS, but are predominantly located in the midline area and in the lateral compartments. The labeled cell bodies are variable in shape and size (largest diameter ranging from 10-30 microns), while both soma and the associated dendritic processes are densely stained for mAChRs. Astrocytes immunoreactive for mAChRs were frequently found associated with the large blood vessels in the midline area. To study the neurotransmitter nature of the mAChR-positive cells, immunofluorescence double-labeling experiments were performed for mAChRs and GABAergic and cholinergic markers. GABAergic cells were identified immunocytochemically using antisera against glutamic acid decarboxylase (GAD), parvalbumin (PARV) or calbindin protein (CaBP). The cholinergic transmitter nature of the mAChR-positive cells was studied using adjacent 8 microns thick serial sections stained immunocytochemically for choline acetyltransferase (ChAT), or histochemically for acetylcholinesterase (AChE). These experiments showed that approximately half (52.3%) of all mAChR-positive cells contain GAD, whereas the other half is cholinergic. Conversely, nearly all GABAergic (98.6%) and cholinergic (96.9%) cells are endowed with mAChRs. GAD-positive terminals were found surrounding mAChR-positive perikarya which were either GAD-positive or GAD-negative, indicating GABAergic innervation on both GABAergic and cholinergic MS neurons. In general, the staining intensity for mAChRs appeared to be considerably higher in GABAergic than in cholinergic neurons, suggesting a stronger cholinergic impact upon the GABAergic neurons. The current anatomical findings contribute to the concept that the MS neurons form a firmly interconnected cell group, in which cholinergic neurotransmission mediated through mAChRs seems to play a significant role.