M 2 muscarinic receptor extracted from Sf9 cells in cholate–NaCl differs from that extracted from porcine sarcolemma. The latter has been shown to exhibit an anomalous pattern in which the capacity for N-[ 3H]methylscopolamine (NMS) is only 50% of that for [ 3H]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [ 3H]QNB. The effects can be explained in terms of cooperativity within a receptor that is at least tetravalent [Park PS, Sum CS, Pawagi AB, Wells JW. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Biochemistry 2002;41:5588–604]. In contrast, M 2 receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [ 3H]NMS at either 30 or 4 °C, although there was a time-dependent inactivation during incubation with [ 3H]NMS at 30 °C; also, any discrepancies in the affinity of NMS were comparatively small. The level of cholesterol in Sf9 membranes was only 4% of that in sarcolemmal membranes, and it was increased to about 100% by means of cholesterol-methyl-β-cyclodextrin. M 2 receptors extracted from treated Sf9 membranes were stable at 30 and 4 °C and resembled those from heart. Cholesterol induced a marked heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [ 3H]NMS, and there were significant discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [ 3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites. Cholesterol therefore appears to promote cooperativity in the binding of antagonists to the M 2 muscarinic receptor.