L-type Ca(2+) channels play a critical role in regulating Ca(2+)-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type Ca(2+) channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type Ca(2+) channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for beta2-adrenergic receptor (AR) regulation of ICa,L. Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel (Cav1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Cav1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Cav1.2, beta2-AR (not beta1-AR), G protein alphas, adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized beta2-AR/Cav1.2 signaling complex, beta2-AR stimulation (salbutamol plus atenolol) of ICa,L was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of ICa,L in response to beta2-AR activation was eliminated by disruption of caveolae with 10 mM methyl beta-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas beta1-AR stimulation (norepinephrine plus prazosin) of ICa,L was not altered. These findings demonstrate that subcellular localization of L-type Ca(2+) channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways.