The beta(1)-adrenoceptor "two-site ligand binding hypothesis" was investigated by comparing the pharmacological activities of the receptor with an Asp to Glu mutation of amino acid 138 after transient transfection into CHO cells. The high-affinity binding of (-)-[(3)H]-CGP12177 (p K(D)=9.4) and binding inhibition by (-)-isoprenaline (p K(i)=6.2), observed with Asp138-beta(1)-adrenoceptors, were absent at Glu138-beta(1)-adrenoceptors. (-)-[(3)H]-CGP12177 bound with a p K(D)=7.6 to Glu138-beta(1)-adrenoceptors and (-)-isoprenaline enhanced binding, probably allosterically. Glu138-beta(1)-adrenoceptors compared with Asp138-beta(1)-adrenoceptors showed a 500,000-fold decrease in cyclic AMP-enhancing potency by (-)-isoprenaline and antagonism by (-)-bupranolol (1 microM) was abolished. At Glu138-beta(1)-adrenoceptors, the agonist potency of (-)-CGP12177, compared with (-)-isoprenaline was reduced five-fold, but the antagonism by (-)-bupranolol (p K(B)=7.1) was not significantly changed, compared with Asp138-beta(1)-adrenoceptor. Thus, Asp138 of the beta(1)-adrenoceptor is essential for the binding of (-)-isoprenaline, (-)-bupranolol and (-)-CGP12177 to a high-affinity site, but not for the binding of (-)-CGP12177 and (-)-bupranolol to a low-affinity site.