SKCa agonists protect against cardiac ischemia and reperfusion injury. SKCa channels are present in cell membranes of vascular and cardiac atrial cells, but not in cell membranes of cardiac ventricular cells. Of the 3 known SKCa isoforms, we found SKCa isoform 3 of splice variant 1 (SKCa 3.1) in the inner mitochondrial (m) membrane of guinea pig ventricular cells. To determine its mitochondrial targeting sequence and function, we cloned full length (FL) and truncated SKCa 3.1 cDNA for overexpression in HL‐1 myocytes. SKCa 3.1 localization was examined by immunocytochemistry and western blotting and channel function was evaluated by K+ uptake into HL‐1 myocyte mitochondria with the K+‐selective fluorescent probe PBFI AM. We found that SK3FL‐EGFP, N‐terminus truncated SK3.1 (SK3Δ1‐277‐EGFP), but not C‐terminus (includes calmodulin binding motif) truncated SK3.1 (SK3Δ626‐720‐EGFP), localized into mitochondria when transfected into HL‐1 cells. Overexpression of SK3FL‐EGFP and SK3Δ1‐277‐EGFP, but not SK3Δ626‐720‐EGFP, increased the rate of K+ uptake in HL‐1 cell mitochondria in response to added KCl and CaCl2. K+ uptake by overexpressed SK3FL‐EGFP was sensitive to the SKCa blocker apamin. Our results show that mSKCa 3.1 requires its C‐terminus not only for mitochondrial targeting but also for Ca2+ ‐stimulated K+ uptake into mitochondria.Grant Funding Source: R01 HL 089514