The β₁- and β₂-adrenergic receptors (βARs) on the surface of cardiomyocytes mediate distinct effects on cardiac function and the development of heart failure by regulating production of the second messenger cyclic adenosine monophosphate (cAMP). The spatial localization in cardiomyocytes of these βARs, which are coupled to heterotrimeric guanine nucleotide–binding proteins (G proteins), and the functional implications of their localization have been unclear. We combined nanoscale live-cell scanning ion conductance and fluorescence resonance energy transfer microscopy techniques and found that, in cardiomyocytes from healthy adult rats and mice, spatially confined β₂AR-induced cAMP signals are localized exclusively to the deep transverse tubules, whereas functional β₁ARs are distributed across the entire cell surface. In cardiomyocytes derived from a rat model of chronic heart failure, β₂ARs were redistributed from the transverse tubules to the cell crest, which led to diffuse receptor-mediated cAMP signaling. Thus, the redistribution of β₂ARs in heart failure changes compartmentation of cAMP and might contribute to the failing myocardial phenotype.