Intermolecular interactions between members of both similar and divergent G protein-coupled receptor subfamilies have been shown in various experimental systems. Here, we demonstrate heterodimerization of predominant β-adrenergic receptor (βAR) subtypes expressed in the heart, β₁AR, and β₂AR, and its physiological relevance. In intact adult-mouse cardiac myocytes lacking native β₁AR and β₂AR, coexpression of both βAR subtypes led to receptor heterodimerization, as evidenced by their coimmunoprecipitation, colocalization at optical resolution, and markedly increased binding affinity for subtype-selective ligands. As a result, the dose-response curve of myocyte contraction to βAR agonist stimulation with isoproterenol (ISO) was shifted leftward by ≈1.5 orders of magnitude, and the response of cellular cAMP formation to ISO was enhanced concomitantly, indicating that intermolecular interactions of βAR subtypes resulted in sensitization of these receptors in response to agonist stimulation. In contrast, the presence of β₁AR greatly suppressed ligand-independent spontaneous activity of coexisting β₂ARs. Thus, heterodimerization of β₁AR and β₂AR in intact cardiac myocytes creates a novel population of βARs with distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation while silencing ligand-independent receptor activation, thereby optimizing β-adrenergic modulation of cardiac contractility.