Incubation of cultured embryonic chicken heart cells with the beta-adrenergic agonist isoproterenol resulted in a dose-dependent increase in the number of mAChR on the surface of intact cells. The isoproterenol-mediated increase in mAChR number was time dependent and reached a maximum by 48 h. Chick heart cells treated with isoproterenol exhibited a greater than 6-fold increase in the sensitivity for carbachol-mediated inhibition of adenylyl cyclase activity as compared to control. Stimulation of cultured heart cells for 24 h with isoproterenol resulted in a 25-35% increase in cm2 mRNA levels as compared to control cm2 mRNA levels. In contrast, the level of cm4 mRNA was not significantly affected by isoproterenol treatment. cm2 mRNA levels were maximally elevated by 15 h following isoproterenol stimulation and remained elevated for up to 72 h. Incubation of cells with isoproterenol in the presence of Rp-cAMP, an inhibitor of cAMP-dependent protein kinase, blocked the increase in the level of cm2 mRNA. Thus, prolonged activation of beta-adrenergic receptors results in an increase in mAChR number and muscarinic responsiveness in chick heart cells due to a cAMP-dependent protein kinase mediated increase in cm2 mRNA levels.