Molecular cloning studies have demonstrated the existence in mammals of five genes encoding distinct muscarinic acetylcholine receptors which are heterogeneously expressed in the central nervous system and the target organs of the autonomic nervous system. In order to determine the factors responsible for regulation of receptor expression, we isolated a genomic clone encoding the mouse m4 muscarinic acetylcholine receptor. The gene contains no introns in the coding sequence, and encodes a polypeptide of 479 amino acids that is able to bind various muscarinic ligands with affinities typical for mAChRs and to couple efficiently to inhibition of adenylyl cyclase.