The properties of muscarinic acetylcholine receptors in the cell line MCM1, derived from an SV40 T-antigen-induced atrial tumor in a transgenic mouse, were determined. Binding studies using the nonselective muscarinic antagonist [3H]quinuclidinyl benzilate, the M1-selective antagonist pirenzepine, and the M2-selective antagonist AFDX-116 indicate that the receptors have the pharmacological properties of the cardiac (M2) receptor subtype. The receptors could be immunoprecipitated with a monoclonal antibody specific for the cardiac receptor, thus confirming the identity of the receptors expressed in these cells. The types of G proteins expressed in the cells were determined by Northern blot analyses: mRNA encoding the alpha subunits of Gs, G(o), and Gi-2, but not Gi-1 or Gi-3, were detected, consistent with previous observations of neonatal mammalian atria. The muscarinic receptors were functionally active, as demonstrated by the ability of the agonist to stimulate phosphoinositide turnover and to inhibit adenylyl cyclase activity. The availability of a mammalian atrial cell line that continues to express the appropriate functionally coupled subtype of muscarinic receptor may provide a useful system for the investigation of the regulation of expression and function of cardiac muscarinic receptors.