We have used the chick embryonic heart to study the regulation of the muscarinic acetylcholine receptor in vivo. Sustained activation of the muscarinic receptor in vivo with the cholinergic agonist carbachol decreases muscarinic receptor number as much as 87% as measured by the specific binding of the potent muscarinic receptor in antagonist [3H]quinuclidinyl benzilate. The decrease in receptor number is both dose and time dependent. After carbachol-induced down regulation, the receptor number recovers to control levels when further receptor activation is blocked by the muscarinic receptor antagonist atropine. When receptor number is decreased 50% in vivo, isolated atria require a 12-fold greater concentration of carbachol than controls to arrest spontaneous beating in an organ bath. Analysis of the binding of carbachol to the muscarinic receptor indicates that this shift in the dose response of atria is accompanied by a change in the relative fraction of the high and low affinity forms of the muscarinic receptor with no change in their respective affinities for carbachol. In addition, this analysis suggests that the low agonist affinity form of the cardiac muscarinic receptor is the physiologically active form.