Competition binding isotherms for agonists to G protein-coupled receptors (GPCR) display high and low binding affinities. Mutagenesis of lysine at position 324 in helix 6 of the wild-type (WT) human beta1-adrenergic receptor (beta1-AR) generated mutant receptors that had GTP-insensitive single low-affinity binding sites for agonists and reduced potencies of full or partial agonists in stimulating adenylyl cyclase. Unlike the WT beta1-AR, intrinsic activities of full and partial agonists in activating the Lys324-->Ala beta1-AR (K324A) mutant were correlated with their binding affinities to the K324A mutant. In assays, such as agonist-mediated phosphorylation and recycling, the K324A mutant and the WT beta1-AR behaved similarly. However, in fluorescence resonance energy transfer assays that determined the proximity between the WT beta1-AR or the K324A mutant to G(s)alpha, there were significant differences. The conceptual framework of the ternary complex model could not adequately account for the behavior of the K324A mutant except under assumptions of low receptor-G protein binding affinities. The single low-affinity binding site of the K324A mutant to isoproterenol was converted by the C-terminal 11-amino-acid peptide of G(s)alpha, which acts a GDP-bound G(s)alpha mimic, to high- and low-affinity sites. Based upon the three-dimensional architecture of the human beta1-AR, the distance between Lys324 and the Asp/Glu-Arg-Tyr motif in helix 3 was the shortest among the various amino acids in helix 6. These findings indicate that Lys324 lies in a groove between helices 3 and 6, and its mutagenesis generates a mutant receptor with very low binding affinity for the GDP-bound isoform of G(s).