Studies with 4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one hydrochloride (CGP 12177) at the human ␤1-adrenoceptor have provided evidence for two binding modes or conformations that have markedly different pharmacological properties. Here, key transmembrane residues (Asp104, Asp138, Ser228, Ser229, Ser232, Phe341, Asn344 and Asn363) have been mutated to provide structural insights into the nature of these conformations. [3 H]CGP 12177 binding and cAMP response element-mediated reporter gene studies confirmed that CGP 12177 was a neutral antagonist (log K D ϭ Ϫ9.18) at the "catecholamine site" and an agonist at the "CGP 12177 site" (log EC 50 ϭ Ϫ8.12). Agonist responses to isoprenaline and CGP 12177 had different sensitivities to ␤1-antagonists (e.g., CGP 20712A; log K D ϭ Ϫ8.65 and Ϫ7.26, respectively). Site-directed mutagenesis showed that Asn363 and Asp138 were key residues for binding of agonists and antagonists, and they were also essential for the agonist actions of CGP 12177. S228A and S229A in transmembranespanning region (TM) 5 reduced the binding of CGP 12177 and had an identical effect on its agonist and antagonist actions. Both N344A and F341A in TM6 abolished the ability of CGP 20712A to discriminate between responses elicited by isoprenaline and CGP 12177. The fact that both Asp138 and Asn363 are absolutely required for CGP 12117 binding in both agonist and antagonist modes leads to the conclusion that the secondary agonist binding site for CGP 12117 must overlap with the catecholamine binding site. Modeling studies provide a basis for these overlapping sites with either the tert-butylamino group or the hydroxyethyloxy and imidazolone portions of CGP 12177 capable of forming polar interactions with Asp138 and Asn363.The ␤1 and ␤2-adrenoceptors are classic examples of G protein-coupled receptors (GPCRs) that couple to G␣ S G-proteins and stimulate adenylyl cyclase activity (Kobilka, 2007). Recent studies of the human and rodent ␤1-adrenoceptors, however, have shown that this receptor exists in at least two conformations that have markedly different pharmacological properties (Granneman, 2001). The initial evidence for multiple binding conformations on the ␤1-adrenoceptor came from detailed studies with the aryloxypropanolamine CGP 12177 (Pak and Fishman, 1996). This compound is a high-affinity neutral antagonist of the classic "catecholamine" binding site or conformation of the ␤1-adrenoceptor; at higher concentrations, however, it activates a secondary site or conformation and produces an agonist response that is relatively resistant to antagonism by other classic ␤-antagonists such as CGP 20712A and propranolol (Pak and Fishman, 1996;Konkar et al., 2000;Baker et al., 2003).Many of our thoughts concerning the specific sites of interaction of ligands with the ␤-adrenoceptors have come from site-directed mutagenesis studies of the ␤2-adrenoceptor. The main binding conformations for catecholamines at the ␤2-adrenoceptor have been identified as Asp113 in tra...