Muscarinic acetylcholine receptors (mAChRs) modulate the activity of an extraordinarily large number of physiological functions. Individual members of the mAChR family (M(1)-M(5)) are expressed in a complex, overlapping fashion in most tissues and cell types. However, the identification of the precise physiological roles of individual mAChR subtypes remains a challenging task because, with the exception of a few snake toxins, mAChR ligands that can activate or inhibit specific mAChR subtypes with a high degree of selectivity are not yet available. Knowledge of the specific roles of mAChR subtypes is of considerable interest for the development of novel, clinically useful mAChR ligands. In this article, recent studies of mutant mouse strains developed, using gene targeting techniques, to be deficient in one of the three G(q)-coupled mAChR subtypes (M(1), M(3) and M(5)) are discussed. These investigations have led to many important new insights into the physiological roles of these receptor subtypes.