The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signalling. In cardiac muscle, caveolae concentrate signalling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation-contraction coupling and consequently cardiac contractility. Evidence from non-cardiac cells has shown that there are multiple caveolar subpopulations, defined by different content of cholesterol, caveolin isoforms, signal effectors (e.g. kinases) and targets (e.g. ion channels). The aim of this investigation was to identify caveolar proteins in cardiac muscle, investigate dynamic regulation of caveolar content, and define co-localised subpopulations of caveolar proteins. We defined the cardiac caveolar proteome using quantitative proteomics to identify proteins depleted from caveolar membranes prepared from rat ventricular myocytes using a standard discontinuous sucrose gradient after treatment of these myocytes with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae. We defined 249 proteins as high-confidence caveolar residents. Functional annotation clustering indicates cardiac caveolae are enriched in integrin signalling, guanine nucleotide binding, ion transport, and insulin signalling clusters. In order to investigate dynamic changes in caveolar protein constituents following adrenoceptor (AR) stimulation we selectively activated α-, β1- and β2-AR by applying agonist/antagonist pairs for 10 min to field-stimulated myocytes prior to preparation of caveolae. Quantitative proteomic analysis indicates that with the notable exception of cavins 1, 2 and 4, very few proteins show altered abundance in caveolae following AR activation, suggesting signalling complexes are pre-formed to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle. To define subpopulations of cardiomyocyte caveolae we enriched caveolar membranes using a sucrose gradient. Membranes were fractionated by size exclusion chromatography using Sephacryl S500 and S1000 columns, and fractions blotted for caveolin1, caveolin3, Na pump a1 subunit and cavin1. Preliminary results show that caveolin1 and caveolin 3 are not enriched in the same fractions, suggesting a subset of cardiomyocyte caveolae is caveolin 1-free. We find cavin1 is associated preferentially with Caveolin 3 over Caveolin 1/3-enriched membranes. This confirms that myocyte caveolae exist in different subpopulations. Hence physical and functional co-localisation of subpopulations of caveolar residents may contribute to the complexity of signalling through these microdomains in cardiac muscle.