Muscarinic acetylcholine receptors (mAChRs; M₁–M₅) play key roles in regulating the activity of many important functions of the central and peripheral nervous system. Because of the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proven a difficult task. To circumvent these difficulties, several laboratories recently employed gene-targeting techniques to generate mutant mouse strains deficient in each of the five mAChR subtypes. Phenotyping studies showed that each mutant mouse line displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits. The novel insights gained from these studies should prove instrumental for the development of novel classes of muscarinic drugs.