The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signalling. Oligomers of caveolin 3 form caveolae within the cardiac sarcolemma. These lipid rafts concentrate many signalling molecules, including G proteins, facilitating cellular signal transduction. The presence of a caveolin binding motif (CBM) is proposed to localise proteins to caveolae via a protein-protein interaction with a scaffolding domain in caveolin. 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. Proteins possessing a CBM were poorly enriched in cardiac caveolae, suggesting this is not the only mechanism that targets proteins to caveolae. Indeed, the overlap between the cardiac caveolar proteome and cardiac palmitoyl proteome suggests that palmitoylation is a more powerful predictor of caveolar localisation that the presence of a CBM, and hence that palmitoylation may recruit proteins to caveolae. To investigate dynamic changes in caveolar protein constituents following adrenoceptor (AR) stimulation we selectively activated α-, β1- and β2-AR 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. Protein glutathionylation (the reversible conjugation of glutathione to protein cysteines in a mixed disulfide) is emerging as a critical signalling event in the cardiovascular system due to its ability to regulate many physiological processes involved in cardiac homeostasis. The heterotrimeric G-protein alpha subunits Gαs and Gαi and caveolin 3 are all glutathionylated (measured using biotinylated glutathione ethyl ester labelling) in unstimulated ventricular myocytes at rest. Treatment of H9c2 cells or ventricular myocytes with the selective thiol oxidizing agent diamide increases glutathionylation of Gαi and caveolin 3. Concurrently co-immunoprecipitation experiments indicate the physical interaction between both Gαi and Gαs and caveolin 3 is lost, however sucrose gradient fractionation indicates both G-protein α subunits still reside within caveolar membranes. This implies that G protein or caveolin 3 glutathionylation dynamically regulates G protein α subunit interaction with caveolin 3 and thus G-protein coupled generation of intracellular cAMP (and consequently contractility) in cardiac muscle. Hence the caveolar signalling microdomain exhibits plasticity in both its composition and molecular interactions. Understanding the basis of this plasticity will further our understanding of caveolar control of cardiac function in both health and disease.