We have determined whether the process of agonist-mediated phosphorylation of the muscarinic receptor correlates with the process of muscarinic receptor desensitization in chick cardiac tissue. Exposure of ventricular slices to the agonist carbachol under conditions previously shown to lead to large increases in muscarinic receptor phosphorylation (Kwatra, M. M., and Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432) resulted in decreased affinity of the muscarinic receptor for agonists. The agonist oxotremorine mimicked and the antagonist atropine prevented the effects of carbachol on receptor phosphorylation and agonist affinity. The time courses and concentration dependences for agonists to induce phosphorylation of the muscarinic receptor and decreases in agonist affinity were similar. Treatment of chick atria with acetylcholine under conditions which led to receptor phosphorylation resulted in decreased sensitivity of these preparations to the negative inotropic effect of carbachol. Taken together, the results support the concept that phosphorylation of cardiac muscarinic receptors may be related to the process of receptor desensitization. The mechanism by which agonists induce receptor phosphorylation was also investigated. The phosphorylated amino acids formed in response to agonists were serine and threonine. The protein kinase C activator phorbol myristate acetate had no effect on receptor phosphorylation or agonist affinity, nor did it prevent the effects of carbachol on either of these parameters. Receptor phosphorylation also was unaffected by the calmodulin antagonists W-7 and W-13, by elevation of cyclic nucleotides, and by agonists which activate other cardiac receptor systems. The results suggest that the phosphorylation of cardiac muscarinic receptors requires agonist occupancy of the receptor and/or may involve the participation of a selective protein kinase.