Caveolae are specialized membrane microdomains enriched in cholesterol and sphingolipids which are present in multiple cell types including smooth muscle cells and cardiomyocytes. Along with the essential scaffolding protein caveolin-3 (Cav-3), a number of different ion channels and transporters have been localized to caveolae. The importance of caveolar localization and regulation of these ion channels is indicated by the finding that mutations in the caveolin-3 gene (CAV3) can cause the congenital long QT syndrome (LQT9). Previous studies have demonstrated that a subpopulation of L-type Ca (Cav1.2) channels is localized to caveolae as part of a macromolecular signaling complex including components of the β-AR/PKA signaling pathway. This localized signaling complex is specifically responsible for β2-AR regulation of cardiac Cav1.2 channels. However, the precise macromolecular composition of Cav1.2 channels present in caveolae and mechanisms targeting these channels to caveolae are not understood. Because the auxiliary Ca channel β subunits (Cavβ1-Cavβ4) influence the trafficking and function of Cav1.2 channels, the present study examines the role of Cavβ subunits in the localization and regulation of caveolar Cav1.2 channels. Immunogold labeling and electron microscopy demonstrated that Cavβ2c but not Cavβ3 co-localized with Cav-3 in ventricular myocytes. GST-Cav-3 pulldown experiments using various Cav-3 domain fusion proteins confirmed that Cav-3 directly associates with Cavβ2 subunit but not with Cavβ1, Cavβ3, or Cavβ4. Immunoprecipitation experiments from transfected HEK293 cells demonstrated that Cav-3 co-immunoprecipitate with Cav1.2 subunit when coexpressed with Cavβ2c subunit. However, Cav1.2 did not co-IP with Cav-3 when Cav1.2 was coexpressed with either Cavβ1b, Cavβ3, Cavβ4 or Cav1.2 subunit alone in HEK293 cells, suggesting Cavβ2c specifically enables caveolar targeting of Cav1.2 channels. The functional role of Cavβ2c subunit on caveolar Cav1.2 channels was analyzed by patch-clamp technique in neonatal mouse cardiomyocytes transfected with either a control siRNA or siRNA specific to Cavβ2c. In the control siRNA transfected myocytes both β1-AR (norepinephrine, 10uM, prazosin, 10uM) and β2-AR specific (salbutamol, 1uM, atenelol 1uM) stimulation significantly increased ICa,L by 100% and 60% respectively. However, when the Cavβ2c subunit expression was knocked down by specific Cavβ2c siRNA transfection, the β2-AR specific stimulation of ICa,L was abolished, where as β1-AR stimulation of ICa,L was intact. siRNA-mediated knockdown of Cavβ2c subunit was confirmed by immunostaining and confocal microscopy. We conclude that the Cavβ2c subunit is an essential component of the caveolar Cav1.2 macromolecular complex and required for β2-AR regulation of Cav1.2 channels.