Studies of beta-adrenergic receptors on several types of intact target cells indicate that agonists, but not antagonists, show prominent KD/Kact discrepancies--lower affinities (KD) in equilibrium binding studies (i.e. in competition with antagonist radioligands) than in functional assays (Kact). In this report we show that intact S49 lymphoma cells initially bind beta-adrenergic agonists in a high affinity manner but that this high affinity binding rapidly converts to a low affinity state. This time course is similar to that of agonist-mediated desensitization in S49 cells. In competitive binding studies conducted with [125I]iodocyanopindolol or [125I]iodohydroxybenzylpindolol, IC50 values for beta-adrenergic agonists increased 13-200-fold between 1 and 60 min, whereas antagonists yielded similar IC50 values at the two time points. The binding of antagonists, but not agonists, could be simulated by a computer model based on the law of mass action. In contrast with results in intact S49 cells, crude membrane fractions yielded similar IC50 values for agonists in 1- and 60-min incubations with [125I]iodocyanopindolol. Moreover, time-dependent decreases in apparent affinity of the agonist (-)-isoproterenol were observed not only in wild type S49 cells but also in several S49 variants (UNC, cyc-, H21a) having defects in Ns, the guanine nucleotide binding protein that couples receptors to activation of adenylate cyclase, and in Kin-, an S49 variant with absent cAMP-dependent protein kinase activity. These results show that beta-adrenergic receptors of intact S49 cells demonstrate a prominent time-dependent decrease in apparent affinity for agonists and that this decrease in affinity does not require cAMP generation, the Ns components defective in several S49 variants or cAMP-dependent protein kinase. The rapid conversion of agonist binding from high to low affinity can account for the rapid desensitization of intact cells to catecholamines and probably explains previously reported KD/Kact discrepancies of intact cells.