Abstract Body: Cardiac hypertrophy, the enlargement of the heart in response to increased workload, is an initially adaptive process that often precedes maladaptive remodeling and heart failure. S-palmitoylation is a reversible post-translational lipid modification of cysteine residues that dynamically regulates the subcellular localization and signaling activity of substrate proteins. Here, we assessed how palmitoylation of the small GTPase Rac1 regulates cardiomyocyte signaling and pathologic cardiac remodeling. Previous literature supports Rac1 as a pro-hypertrophic signaling molecule as cardiomyocyte-specific deletion of Rac1 in mice protects against both angiotensin II (AngII) or pressure overload-induced cardiac hypertrophy. We found that blunting Rac1 palmitoylation by mutation of cysteine-178 to serine resulted in reduced steady-state Rac1 activity in cardiomyocytes. To assess the functions of Rac1 palmitoylation in cardiac pathophysiology, we generated conditional knock-in (Rac1^cKI) mice which have cardiomyocyte-specific expression of palmitoylation-deficient Rac1^C178S. We found that Rac1^cKI mice display normal cardiac structure and function with age. However, in response to three separate models of hypertrophic stress (AngII infusion, pressure overload, or transgenic AT1R overexpression), Rac1^cKI mice develop more severe cardiac hypertrophy and functional decompensation compared to control mice. We found that hearts from Rac1^cKI mice, but not control mice, subjected to any form of hypertrophic stress displayed increased protein kinase A (PKA) substrate phosphorylation and subsequent experiments showed that Rac1^cKI myocytes are hypersensitive to isoproterenol stimulation, suggesting impaired response to adrenergic drive may be partly responsible for the maladaptive remodeling observed in these mice. Rac1^cKI mice also have reduced levels of Ppp2r3a/PR72, a regulatory subunit of the protein phosphatase 2A (PP2A) complex that negatively regulates PKA signaling in cardiomyocytes. Ongoing studies are investigating how palmitoylation of Rac1 regulates PKA/PP2A signaling in response to beta-adrenergic stimulation and hypertrophic stress.