The beta-adrenergic antagonist, [3H]dihydroalprenolol, was used to label binding sites in crude rat myocardial plasma membranes. The specificity of binding was dependent on the temperature of the assay. Specific binding at 22 and 37 degree C and at concentrations of radioligand less than 5 nM was consistent with binding to the myocardial beta-receptor. Binding sites labeled at 4 degree C possessed quite different properties. Binding was non-stereoselective and of lower affinity. Agonist compounds were much less effective at competing for the labeled myocardial sites at 4 degree C than at 22 degree C. Those beta-antagonists which additionally possess pharmacological "quinidine-like' activity (e.g. propranolol, alprenolol) were potent competitors at 4 degree C, but competition was non-stereoselective. In contrast atenolol, a beta-antagonist devoid of "quinidine-like' activity was ineffective at 4 degree C. Furthermore, procaine, and quinidine itself were potent competitors of [3H]dihydroalprenolol binding at 4 degree C. Thus the specificity of [3H]dihydroalprenolol binding to rat heart membranes at 4 degree C appears to be directed non-stereoselectively at that portion of the competing molecule which recognized "quinidine-like' as opposed to adrenergic activity.