[125I]Iodohydroxybenzylpindolol, an extremely potent beta-adrenergic antagonist, has been purified to theoretical specific activity (2200 Ci/mmol) and used as a ligand to characterize the beta-adrenergic receptors of cultured rat glioma cells and other cultured cell clones. Appropriate receptor sites were identified by stereoselective competition for binding by a number of adrenergic agonists and antagonists, by correlation between the potency of these compounds to inhibit binding and to affect adenylate cyclase activity, and by correlation of binding with the presence or absence of response to catecholamines (stimulation of adenylate cyclase) in various cell clones. In equilibrium experiments, the dissociation constant for binding of the iodinated ligand to the beta-adrenergic receptor of a clone of rat glioma cells (C6TG1A) was 250 pM; the corresponding value for a clone of human fibroblasts (VA2) was 15 pM. For C6TG1A, KD was verified by analysis of the kinetics of binding: k1 = 10(8) 1/mol/min: k-1 = 0.017/min. This rate of dissociation of ligand from the receptor was also established by study of the rate of activation of adenylate cyclase by isoproterenol after prior equilibration with iodohydroxybenzylpindolol. For VA2 cells, where affinity was higher, the rate of reversal of binding was only 0.0035/min. C6TG1A contained approximately 4000 receptor sites/cell (75 fmol/mg of protein), and these sites appeared to be coupled to adenylate cyclase in a stoichiometric manner. A second site with equal affinity for iodohydroxybenzylpindolol (KD = 250 pM) was also identified in C6TG1A by both kinetic analysis and equilibrium binding studies. While most compounds that interacted with the beta-adrenergic receptor also influenced binding to the second site, the latter did not distinguish between stereoisomers of propranolol, and its affinity for the other compounds tested was poorer.