The cardiac sodium/calcium exchanger (NCX1) plays a vital role in the regulation of intracellular Ca in cardiac muscle, removing Ca that enters the cell during systole. Inappropriate NCX1 function contributes to cardiac contraction abnormalities and heart failure: reduced NCX1 activity reduces Ca removal and therefore impairs relaxation, whereas overactive NCX1 unloads the intracellular Ca stores and impairs systolic function. Although the structure-function relationship of NCX1 is well characterised, the dynamic regulation of NCX1 function is controversial. The reversible attachment of the 16-carbon fatty acid palmitate to cysteine residues in proteins (palmitoylation) is an important and common post-translational modification in a variety of tissues. However, the cardiac palmitoyl proteome remains largely uncharacterised. In general, palmitoylation targets proteins to the lipid bilayer as addition of palmitate increases the hydrophobicity of proteins. With respect to ion transporters, palmitoylation controls cell surface expression, spatial organisation, and voltage-sensitivity as well as cross talk with other signalling pathways (Linder & Deschenes 2007). Given that multiple ion channels and transporters are palmitoylated, palmitoylation of cardiac calcium handling proteins may be as important as phosphorylation in the regulation of excitation-contraction coupling and thus cardiac output. We investigated the palmitoylation of NCX1 from ventricular myocytes (VM) isolated from adult male Wistar rats by retrograde perfusion of collagenase. Using site-specific resin assisted capture (Acyl Rac), palmitoylated proteins were purified from isolated adult rat VM. Data is presented as mean±SEM, with differences analysed by t-test. NCX1 is substoichiometrically palmitoylated in VM (proportion of NCX1 purified by Acyl Rac: 73±5%, n=6) compared to the constitutively palmitoylated protein, caveolin-3. We expressed NCX1 in HEK-293 cells by transient transfection and measured palmitoylation by Acyl Rac, and cell surface localization using membrane impermeable biotinylation reagents. The global palmitoylation inhibitor 2-bromopalmitate (100μM, 18 hours), significantly reduced NCX1 palmitoylation (by 89±4%% compared to untreated cells, n=4, p<0.001), and significantly decreased cell surface expression of NCX1 (by 49±11% compared to untreated cells, n=3, p<0.05). NCX1 is regulated via a large intracellular loop between transmembrane domains 5 and 6, in which a number of cysteine residues are located. Site-directed mutagenesis of cysteines 383, 387, 485 or 557 did not reduce NCX1 palmitoylation in transiently transfected HEK-293 cells. In conclusion, palmitoylation of NCX1 influences its turnover at the plasma membrane and/or processing in the secretory pathway. The effect of palmitoylation on NCX1 activity is currently under investigation.