We have studied binding parameters (Kd, Bmax) of [3H]N-methylscopolamine ([3H]NMS) in various brain regions and spinal cord of wild-type (WT) and muscarinic acetylcholine receptor (mAChR) subtype (M1-M5) knockout (KO) mice. In the M1-M4 KO mice, the number of [3H]NMS binding sites (Bmax) was decreased throughout the central nervous system (CNS) with significant regional differences. Our results collectively suggest that M1 receptor was present in a relatively high density in the cerebral cortex and hippocampus, and the densities of M1 and M4 subtypes were highest in the corpus striatum. M2 receptor appeared to be the major subtype in the thalamus, hypothalamus, midbrain, pons-medulla, cerebellum and spinal cord. These findings may contribute significantly not only to the further understanding of the physiological roles of mAChR subtypes in the central cholinergic functions, but also to the development of selective therapeutic agents targeting specific subtype.