Acute regulation of the cardiac Na pump by extracellular stimuli is crucial in allowing the heart to match its output to systemic requirements. The Na pump not only plays a critical role in a plethora of transmembrane transport process, electrical excitability and cell volume control but also, by setting intracellular Na, is a primary determinant of cardiac contractility because it determines the set-point for the sarcolemmal sodium-calcium exchanger. S-palmitoylation is a reversible covalent post-translational attachment of the fatty acid palmitic acid to the thiol group of cysteine, via an acyl-thioester linkage. Palmitoylation can alter enzymatic / ion channel activity, stability or subcellular localisation of the target protein, and this is usually achieved by the recruitment of the palmitoylated cysteine to the lipid bilayer. As such, palmitoylation has the potential to induce substantial changes in protein secondary structure and thus function, through the recruitment of intracellular loops to the inner surface of the membrane bilayer. We investigated palmitoylation of the catalytic α subunit of the Na pump in ventricular myocytes isolated from adult male Wistar rats. Palmitoylated proteins were purified by resin-assisted capture, and palmitoylation sites identified by differential alkylation of palmitoylated and non-palmitoylated cysteines, followed by analysis by LC-MS/MS against inclusion lists. Cys 374 in the α1 subunit of the Na pump was found palmitoylated. Located within the P (phosphorylation) domain of the α1 subunit which is transiently phosphorylated during the reaction cycle of the pump, C374 lies only 2 amino acids from the central catalytic aspartate (376), the site of this transient phosphorylation, meaning its palmitoylation is highly likely to influence pump activity. In order to determine which acyl palmitoyl transferase (DHHC-PAT) palmitoylates the Na pump α1 subunit, all 25 mammalian DHHC-PATs were individually transiently overexpressed in HEK-293 cells, and palmitoylation of the endogenous Na pump α1 subunit assessed by immunoblotting after resin-assisted capture. Only expression of DHHC15 significantly increased palmitoylation of the α1 subunit (4.6±1.8 fold over mock transfected control, n=5, mean±SEM p<0.05, t-test), implicating this DHHC-PAT in pump α subunit palmitoylation. DHHC15 is a Golgi-localised DHHC-PAT. We therefore propose that α subunit palmitoylation occurs early in the lifetime of the cardiac Na pump complex, and may represent a mechanism to inhibit futile ATP hydrolysis and / or Na-loading of intracellular compartments by the Na pump during it passage through the secretory pathway. The control of α subunit depalmitoylation upon delivery to the surface membrane may therefore represent a crucial step in controlling the number of active Na pumps at the cell surface.