The binding of acetylcholine to receptors in the intact heart causes a decrease in the frequency (chronotropic effect) and force (ionotropic effect) of contraction. The studies reported here demonstrate a chronotropic response of cultured embryonic chicken heart cells to the muscarinic agonist carbamoylcholine. This response is markedly decreased after a 3-hr incubation with 0.1 mM carbamoylcholine. In order to determine whether agonist-induced alterations in muscarinic receptors were responsible for this decrease, we studied the effects of incubation with carbamoylcholine on the binding of the (3)H-labeled muscarinic antagonist quinuclidinyl benzilate (QNB) to homogenates of heart cell cultures. [(3)H]QNB binding to homogenates of cultures of embryonic hearts of chicks 9 days in ovo was characterized and shown to have properties similar to those of muscarinic receptors in intact hearts. Binding was both specific and saturable. [(3)H]QNB was displaced by muscarinic agonists and antagonists in concentrations consistent with their known potency. Binding was poorly inhibited by the nicotinic antagonist D-tubocurarine. Kinetic analysis of the binding of QNB by muscarinic receptors showed that initially the reaction proceeds by formation of a rapidly reversible complex with a K(d) of 1.8 nM, which is converted to a slowly reversible form. These properties of muscarinic receptors in heart cell cultures are strikingly similar to those observed in homogenates of intact hearts. Homogenates of heart cell cultures bound 84 +/- 6 fmol (mean +/- SD) of QNB per mg of protein. The number of receptors remained stable from day 4 to day 8 in culture. Incubation of cultures with 0.1 mM carbamoylcholine for 3 hr decreased QNB binding by 55%, to 38 +/- 5 fmol/mg protein. When cell cultures were first homogenized and then incubated with carbamoylcholine, no decrease in QNB binding sites could be detected. Thus, incubation with carbamoylcholine causes loss of muscarinic binding sites as well as decreased physiologic responsiveness to muscarinic agonists.