The regulation of the muscarinic receptor by the agonist methacholine was investigated in the perfused working rat heart. After 2.5 hr of perfusion with 4 microM methacholine, the number of (-)-[3H]-quinuclidinyl benzilate binding sites was significantly reduced (10-18%) in the right and left atria and left ventricle when compared with controls (2.5 hr of perfusion in the absence of methacholine). Scatchard analysis revealed that there was no change in the apparent dissociation constant for quinuclidinyl benzilate. Furthermore, when hearts were perfused for 2.5 hr with methacholine in the presence of (-)-scopolamine, a muscarinic receptor antagonist, no significant loss of receptor was observed. After perfusing with methacholine for 2.5 hr followed by 2.5 hr in the absence of agonist, the number of quinuclidinyl benzilate binding sites returned to control levels. These results suggest that agonist-induced receptor decreases or down regulation requires receptor activation and not just simple receptor occupancy. This decrease, moreover, is reversible. The perfused working rat heart represents a physiological system in which the mechanisms of muscarinic receptor regulation can be studied.