In the supernatant (30,000 x g) of frog erythrocyte homogenates, by using gel filtration we detected a protein that could bind [(3)H]dihydroalprenolol ([(3)H]DHA) with high affinity. This binding was greatly enhanced when the erythrocytes were preincubated with (-)-isoproterenol. After various periods of incubation with (-)-isoproterenol, the extent of the increase in the density of [(3)H]DHA binding sites in the cytosol was paralleled by a proportional decrease in the number of [(3)H]DHA binding sites in the corresponding pellet; both events peaked after 2-3 hr of incubation with (-)-isoproterenol. The K(a) of the (-)-isoproterenol-induced increase in [(3)H]DHA binding in cytosol and the decrease in this binding in the membrane ranged between 60 and 90 nM. The changes in the cytosol and particulate [(3)H]DHA binding sites were independent of RNA and protein synthesis. The increase in cytosol binding elicited by (-)-isoproterenol was blocked by exposure of the cells to (-)-alprenolol which per se failed to change the cytosol binding of [(3)H]DHA. Scatchard analysis revealed that the enhanced [(3)H]DHA binding to cytosol material was due to a 4-fold increase in the B(max) with little or no change in K(d) ( approximately 9 nM). Binding displacement data show that these soluble [(3)H]DHA binding sites resemble the surface membrane recognition sites. Moreover, the ability of various beta-adrenergic agents to increase [(3)H]DHA binding to cytosol after they were incubated with frog erythrocytes paralleled their affinity for membrane-bound beta receptors. These findings support the view that the beta-adrenergic receptor desensitization caused by prolonged exposure to (-)-isoproterenol is due, at least in part, to an internalization of the recognition site of beta-adrenergic receptors.