Acute regulation of cardiac function requires cellular mechanisms to elicit changes in protein function through different post-translational modifications. One such modification is palmitoylation, the addition of the 16-carbon fatty acid palmitate to a cysteine thiol side chain, which alters protein function by regulating intracellular sorting, stability, membrane interactions, enzyme activity and targeting to lipid rafts. Glutathione acts as a redox buffer and antioxidant within cells in order to protect proteins from irreversible oxidative damage. Protein glutathionylation (the reversible conjugation of glutathione to protein cysteines in a mixed disulfide) is emerging as a critical signaling event in the cardiovascular system due to its ability to regulate many physiological processes involved in cardiac homeostasis. Both palmitoylation and glutathionylation can change the function and location of a protein in a reversible manner; we investigated competition between these modifications in both freshly isolated rat ventricular myocytes and the rat cardiomyoblast cell line H9c2. The heterotrimeric G-protein alpha subunits Gas and Gai and caveolin 3 are all palmitoylated (measured using resin-assisted capture) and glutathionylated (measured using biotinylated glutathione ethyl ester labelling) in unstimulated cells at rest. Oligomers of caveolin 3 form caveolae within the cardiac sarcolemma. These lipid rafts concentrate many signalling molecules, including G proteins, facilitating cellular signal transduction. Treatment of H9c2 cells or ventricular myocytes with the selective thiol oxidizing agent diamide increases glutathionylation of Gai and Gas and caveolin 3 (2.47 ± 0.77, n=7). Concurrently co-immunoprecipitation experiments indicate the physical interaction between both Gai and Gas and caveolin 3 is lost, however sucrose gradient fractionation indicates both G-protein a subunits still reside within caveolar membranes. This implies that G protein or caveolin 3 glutathionylation dynamically regulates G protein a subunit interaction with caveolin 3 and thus G-protein coupled generation of intracellular cAMP. Control of intracellular cAMP is functionally crucial in the regulation of cardiac function. Determining the role of glutathionylation on Gas, Gai and caveolin 3 will therefore provide considerable insight into dynamic regulation of cardiac function both at rest and under conditions of redox stress such as ischemia reperfusion.