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The electrogenic Na/Ca exchanger (NCX) mediates bidirectional Ca movements that are highly sensitive to changes of Na gradients in many cells. NCX1 is implicated in the pathogenesis of heart failure and a number of cardiac arrhythmias. We measured NCX1 palmitoylation using resin-assisted capture, the subcellular location of yellow fluorescent protein-NCX1 fusion proteins, and NCX1 currents using whole-cell voltage clamping. Rat NCX1 is substantially palmitoylated in all tissues examined. Cysteine 739 in the NCX1 large intracellular loop is necessary and sufficient for NCX1 palmitoylation. Palmitoylation of NCX1 occurs in the Golgi and anchors the NCX1 large regulatory intracellular loop to membranes. Surprisingly, palmitoylation does not influence trafficking or localization of NCX1 to surface membranes, nor does it strongly affect the normal forward or reverse transport modes of NCX1. However, exchangers that cannot be palmitoylated do not inactivate normally (leading to substantial activity in conditions when wild-type exchangers are inactive) and do not promote cargo-dependent endocytosis that internalizes 50% of the cell surface following strong G-protein activation or large Ca transients. The palmitoylated cysteine in NCX1 is found in all vertebrate and some invertebrate NCX homologs. Thus, NCX palmitoylation ubiquitously modulates Ca homeostasis and membrane domain function in cells that express NCX proteins.-Reilly, L., Howie, J., Wypijewski, K., Ashford, M. L. J., Hilgemann, D. W., Fuller, W. Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling.