Muscarinic receptors were solubilized from porcine atria in digitonin-cholate and were purified by chromatography on DEAE-Sepharose and 3-(2'-aminobenzhydryloxy)tropane-Sepharose. The product identified on Western blots migrated with an apparent molecular mass of 60-75 kDa, with additional bands indicative of homotrimers (190 kDa) and homotetramers (240 kDa). Receptor eluted from the affinity column was accompanied by a mixture of guanyl nucleotide-binding proteins (G-proteins) identified on Western blots as Gi1/2, G(o), Gq/11, and Gs (preparation M2G); the G-proteins were largely removed by further processing on hydroxyapatite (preparation M2). Solubilized purified receptors bound muscarinic ligands in an apparently cooperative manner. In studies at equilibrium, the antagonists [3H]AF-DX 384, N-[3H]methylscopolamine (NMS), and [3H]quinuclidinylbenzilate (QNB) revealed Hill coefficients between about 0.8 and 1.2. Also, the apparent capacity for [3H]QNB exceeded that for [3H]AF-DX 384 and [3H]NMS by about 1.5-fold in M2 and by 2-fold in M2G. Binding to M2G at high concentrations of [3H]QNB was fully inhibited by unlabeled NMS, which therefore affected sites not labeled at similar concentrations of [3H]NMS. Oxotremorine-M displayed a biphasic inhibitory effect on the binding of [3H]AF-DX 384 in M2 and M2G, suggesting that multiple states of affinity are intrinsic to the receptor; 5'-guanylylimidodiphosphate was without appreciable effect in M2 but resulted in a bell-shaped binding profile for the agonist in M2G. All of the data can be described in terms of cooperative interactions within a receptor that is at least tetravalent and presumably an oligomer. In the context of the model, copurifying G-proteins and guanyl nucleotides serve to regulate the degree of cooperativity between successive equivalents of muscarinic ligands.