(-)-[3H]Dihydroalprenolol, a potent competitive beta-adrenergic antagonist, has been previously documented to bind to the adenylate cyclase-coupled beta-adrenergic receptor sites in mammalian and non-mammalian tissues. Steady state binding of (-)-[3H]dihydroalprenolol to sites in frog erythrocyte membranes, a model system for adenylate cyclase-coupled beta-adrenergic receptors, displays characteristics consistent with negative cooperativity among the beta-adrenergic receptors: Scatchard plots are curvilinear with upward concavity and slopes of Hill plots are consistently less than 1.0. The existence of site-site interactions of the negatively cooperative type were demonstrated directly by the ability of unlabeled (-)-alprenolol to accelerate the dissociation of (-)-[3H]dihydroalprenolol under conditions were no rebinding of radioligand occurred. The dissociation rate of (-)-[3H]dihydroalprenolol alone is directly related to temperature and increases with increases in temperature from 4-37 degrees. (-)-[3H]Dihydroalprenolol dissociation is enhanced by unlabeled (-)-alprenolol at all temperatures studied; however, at 4 degrees, the time required to observe an enhancement of radioligand dissociated is greater than the time required for unlabeled (-)-alprenolol to occupy the empty receptor sites, suggesting that increased rigidity of the biomembrane at 4 degrees may be responsible for the absence of readily observable site-site interactions. The ability of a number of beta-adrenergic agonists and antagonists to induce negative cooperativity among the beta-adrenergic receptors was directly related to their affinity for the receptor sites rather than their intrinsic activity in the adenylate cyclase-coupled beta-adrenergic system. The ability to induce site-site interactions among the beta-adrenergic receptors occurs at physiological concentrations of beta-adrenergic agents, since occupancy of less than 10% of the receptor sites is sufficient to reduce receptor affinity. Changes in pH from 6.5 to 9.0 did not significantly alter the negatively cooperative site-site interactions among the receptor sites. The negatively cooperative phenomenon was also independent of Mg2+, Ca2+, and NaF concentrations in the buffer medium. The presence of guanyl-5'-yl imidodiphosphate, a nonhydrolyzable nucleotide analog which enhances adenylate cyclase stimulation (Vmax) by beta-adrenergic agonists and decreases the concentration of agonist required to half-maximally stimulate adenylate cyclase, did not alter the ability of either agonists or antagonists to induce negatively cooperative site-site interactions among the beta-adrenergic receptors.