Several conformations of muscarinic acetylcholine receptors in chick heart are revealed by the energetics of agonist binding. Heating cardiac membranes at 50 degrees C for 5 min decreased agonist affinity, steepened agonist binding curves and eliminated receptor sensitivity to guanine nucleotides. N-ethylmaleimide (NEM) steepened agonist binding curves and eliminated receptor sensitivity to guanine nucleotides without decreasing agonist binding affinity. NEM prevented and reversed the effect of heat on agonist affinity. Pirenzepine and N-methylscopolamine binding were unaffected by either treatment. NEM and heat stabilize muscarinic receptors in conformations unrelated to those associated with naturally occurring receptor populations.