The purpose of this study was to investigate whether caveolin-3 (Cav3) regulates localization of β2-adrenergic receptor (β2AR) and its cAMP signaling in healthy or failing cardiomyocytes. We co-expressed wildtype Cav3 or its dominant-negative mutant (Cav3DN) together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps in adult rat ventricular myocytes (ARVMs). FRET and scanning ion conductance microscopy were used to locally stimulate β2AR and to measure cytosolic cAMP. Cav3 overexpression increased the number of caveolae and decreased the magnitude of β2AR-cAMP signal. Conversely, Cav3DN expression resulted in an increased β2AR-cAMP response without altering the whole-cell L-type calcium current. Following local stimulation of Cav3DN-expressing ARVMs, β2AR response could only be generated in T-tubules. However, the normally compartmentalized β2AR-cAMP signal became diffuse, similar to the situation observed in heart failure. Finally, overexpression of Cav3 in failing myocytes led to partial β2AR redistribution back into the T-tubules. In conclusion, Cav3 plays a crucial role for the localization of β2AR and compartmentation of β2AR-cAMP signaling to the T-tubules of healthy ARVMs, and overexpression of Cav3 in failing myocytes can partially restore the disrupted localization of these receptors. © 2013 The Authors.