Binding of the lipophilic antagonist ligand 3H-DHA and the hydrophilic antagonist ligand 3H-CGP 12177 to beta-adrenergic receptors of rat heart ventricular membranes was studied. Quantitative analysis of the binding data indicated the existence of two specific affinity states of the beta-adrenergic receptor population aside from a third non-specific binding site for 3H-DHA. In order to exclude that the biphasic saturation isotherm may be due to retained endogenous agonist, crude membranes as well as modified membranes were used. In the latter a 99% reduction of noradrenaline concentration was obtained by washing and preincubation or by catecholamine depletion. Two affinity states of antagonist binding could be demonstrated independently from the kind of membrane suspension. A biphasic dissociation of 3H-DHA by unlabelled (-)-alprenolol was also found in kinetic studies. In crude or washed membranes of untreated rats the guanine nucleotide Gpp(NH)p affected saturation and antagonist competition curves. However, this was not observed in catecholamine-depleted membranes of reserpine-treated rats. Stereoselectivity of the high and the low affinity state was demonstrated in competition experiments with (-) - and (+) -alprenolol in catecholamine-depleted membranes. The data are best explained by assuming a ternary complex model (1) in which antagonists, instead of passively occupying binding sites, play an active role in receptor mechanisms. Based on this model, it is assumed that beta-adrenergic antagonists bind with high affinity to the free form of the receptor and with low affinity to the precoupled form. Furthermore, an interaction of Gpp(NH)p with the regulatory component is proposed.