We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined the developmental regulation of mAChR expression in the chick retina. Early in development, the M4 receptor is the predominant mAChR while the levels of the M2 and M3 receptors increase later in development. The level of M2 receptor is also initially very low in retinal cultures and undergoes a dramatic increase over several days in vitro. The level of M2 receptor can be increased by a potentially novel, developmentally regulated, secreted factor produced by retinal cells. The promoter for the chick M2 receptor gene has been isolated and shown to contain a site for GATA-family transcription factors which is required for high level cardiac expression. The M2 promoter also contains sites which mediate induction of transcription in neural cells by neurally active cytokines. We have generated knockout mice lacking the M1 receptor and shown that these mice do not exhibit pilocarpine-induced seizures and muscarinic agonist-induced suppression of the M-current potassium channel in sympathetic neurons.