The high-affinity agonist binding state of muscarinic receptors in the rat heart and cerebral cortex has been pharmacologically characterized in parallel studies. Muscarinic sites were labeled and studied with the aid of a highly specific, rapid filtration binding assay using the potent muscarinic agonist (+)-[3H]CD. Homogenates of both tissues were found to contain a saturable high-affinity (Kd = 1-2 nM), low capacity (6-17% of (-)-[3H]QNB sites) (+)-[3H]CD binding state which demonstrated stereoselectivity and drug specificity typical of a muscarinic site. However, comparative studies of drug potency profiles in competition for myocardial and cerebral cortical (+)-[3H]CD-labeled membranes revealed several major pharmacological differences between muscarinic sites in these tissues. Whereas the muscarinic agonists pilocarpine and McN-A-343, the nonclassical antagonist pirenzepine, and the acetylcholinesterase inhibitor physostigmine reduced (+)-[3H]CD binding in both tissues, their inhibitory effects were more potent (4- to 77-fold) in cerebral cortical membranes. Conversely, gallamine, a nicotinic cholinergic antagonist, demonstrated a 36-fold greater potency at the high-affinity (+)-[3H]CD binding state in myocardial membranes. By comparison, other classical muscarinic agonists and antagonists were nearly equipotent as inhibitors of high-affinity (+)-[3H]CD binding in these two tissues. Thus, these studies for the first time demonstrate that muscarinic receptors in the heart and cerebral cortex can be distinguished pharmacologically by certain drugs which interfere with the high-affinity agonist binding state of the muscarinic recognition site and provide support for the subclassification of these receptors.