A genomic fragment corresponding to sequences encoding the N terminus of the third transmemebrane region to the C terminus of the third intracellular loop was replaced with a neomycin resistance cassette. In situ hybridization experiments demonstrated that no transcript was detectable in brain tissue of homozygous mice. In addition, mutant hearts were unable to bind significant amounts of the muscarinic radioligand [3H]QNB, and muscarinic agonist-induced bradycardia was no longer observed.