The present investigation has assessed the influence of assay buffer (Tris versus Krebs) on the abilities of several beta-adrenoceptor agonists and antagonists to displace the radioligand (-)-[125I]iodocyanopindolol [( 125I]CYP) from beta 1-(left atrial) and beta 2- (uterine) adrenoceptor sites. Saturation studies indicated that the dissociation constant (KD) for [125I]CYP at both beta-receptor sites was approximately 2-fold greater in Krebs as opposed to Tris buffer, and that the maximal density of binding sites (Bmax) in atria (but not uterus) was also reduced 2-fold. In general, the KD values for beta-adrenoceptor agonists were more influenced by the type of buffer used than were KD values for antagonists. Agonist KD values at beta 2-adrenoceptor sites were higher in Krebs than in Tris buffer, while at beta 1-adrenoceptor sites, variable changes resulted. The selective affinity of agonists at beta 1- and beta 2-adrenoceptor sites was therefore markedly influenced by the buffer used and could not be predicted from the established beta 1-/beta 2-adrenoceptor selectivity of the agonists as found in organ bath studies.