Publisher Summary This chapter discusses the phenotypical analysis and clinical implications of muscarinic acetylcholine (ACh) receptor knockout mice. Molecular cloning studies have revealed the existence of five molecularly distinct muscarinic acetylcholine (ACh) receptor (mAChR) subtypes (M1–M5). At a molecular level, the M1, M3, and M5 receptors preferentially couple to G proteins of the Gq/G11 family, whereas the M2 and M4 receptors are primarily linked to G proteins of the Gi/Go class. Studies with mAChR agonists and antagonists have shown that mAChRs are involved in the control of numerous fundamental physiological processes. Central mAChRs are known to regulate a large number of vegetative, sensory, and motor functions. Moreover, central muscarinic mechanisms play important roles in arousal, attention, rapid eye movement (REM) sleep, emotional responses, stress modulation, and higher cognitive processes such as memory and learning. The chapter reviews the major phenotypes displayed by mutant mice lacking M2, M3, or M4 mAChRs. The M2 and M4 mAChR genes were inactivated via homologous recombination in mouse embryonic stem (ES) cells. Homozygous M2-/- (M2R-/-) and M4-/- receptor (M4R-/-) mutant mice were obtained with the expected Mendelian frequency, indicating that there was no increase in embryonic or postnatal mortality. Moreover, wild-type (WT) and M2 and M4 receptor mutant mice did not differ in overall health, were fertile, and bred normally.