The major focus of this paper is the characterization and quantification of rat cardiomyocyte, cell-surface beta-adrenergic receptors labelled with the hydrophilic radioligand [3H]CGP-12177. The ventricular cardiomyocytes used in these experiments have previously been extensively studied in our laboratory and confirmed to be functionally compatible with similar cells in vivo. Specific binding of [3H]CGP was stereospecific, saturable and of high affinity. Binding of [3H]CGP was also readily reversible, demonstrated appropriate drug specificity and positively correlated with increasing cell concentrations. The potency of the beta 1-antagonist atenolol was almost 100 times higher than that of the beta 2-antagonist ICI-118.551 in binding to the [3H]CGP binding site. This preparation appears ideal for the investigation of beta-adrenergic receptor regulation in heart cells. Indeed, our initial experiments show clearly that pharmacological concentrations of isoproterenol, and norepinephrine, can reduce (down-regulate) the number of specific [3H]CGP binding sites. This result is in agreement with many other reports on similar experiments in a variety of cell types. However, physiologically relevant concentrations of these two agonists (1-100 nM) do not induce down-regulation of the beta-adrenergic receptors in short-term (2 h) incubations. Nevertheless, the high-affinity receptors that we have described mediate a contractile response to isoproterenol in the nanomolar concentration range (EC50 = 3.6 +/- 0.3 nM). This is approximately 300 times lower than the concentration needed to produce down-regulation. Thus, our data indicate that short-term down-regulation of cardiomyocyte beta-adrenergic receptors can only be observed with high, pharmacological concentrations of isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)