Cooperativity has been investigated as the mechanistic basis for effects observed with cardiac muscarinic receptors in washed membranes from Syrian hamsters. Specifically, N-[3H]methylscopolamine labeled only 66-75% of the sites labeled by [3H]quinuclidinylbenzilate at apparently saturating concentrations of each radioligand. Also, receptors labeled by N-[3H]methylscopolamine revealed three states of affinity for agonists, both in native membranes and following irreversible blockade of about 80% of the sites by propylbenzilylcholine mustard; in both preparations, guanylylimidodiphosphate (GMP-PNP) effected an apparent interconversion of sites from higher to lower affinity for agonists and from lower to higher affinity for the antagonist. Excellent and mechanistically consistent descriptions of the data were obtained in terms of a model comprising cooperative and noncooperative forms of the receptor; the former was described by a variant of the Adair equation, and the latter was included to account for low-affinity sites that survived treatment with the mustard. If differences in apparent capacity derive from negative cooperativity in the binding of N-[3H]methylscopolamine, the cooperative form of the receptor was at least trivalent in native membranes; otherwise, constraints imposed by the effects of GMP-PNP at the concentrations of radioligand used in the assays dictate that the cooperative form of the receptor was at least tetravalent. In contrast, a divalent receptor is sufficient with the data from alkylated membranes, in accord with the reduced likelihood of interactions between functional sites within an oligomeric array. A model is presented wherein the receptor interconverts spontaneously between two or more states differing in their cooperative properties. The effects of GMP-PNP can be rationalized as a shift in the equilibrium between the different states.