Our previous immunochemical and transcript analyses indicate that the large conductance calcium-activated potassium channel (BKCa) is present in cardiac mitochondria and encoded by the Kcnma1 gene. In addition, studies using a BKCa opener, NS1619, point to the view that opening BKCa expressed in mitochondria (mitoBKCa) contributes to cardioprotection after ischemic insult bymodulating mitochondrial function. However, whether the beneficial effect of NS1619 depends on the presence of Kcnma1 has not been studied. To address this question, we used mice expressing (wild type, wt) or not (Kcnma1-/-) Kcnma1 gene product and the Langendorff model of global ischemia and reperfusion.Mitochondrial functions affecting the opening of the mitochondrial permeability transition pore (mPTP) and cell death were assessed: calcium retention capacity (CRC) and reactive oxygen species (ROS). Mitochondria were isolated after 10 min of reperfusion from hearts preconditioned or not (control) for 10 min before ischemia with 10 μM NS1619. Mitochondria from hearts preconditioned with NS1619 showed a significant increase in CRC from 140+11 to 280+23 nmoles/mg (n=3) in wt mice but no significant effect was observed in Kcnma1-/- mice (120+23 and 100+11 nmoles/mg, n=3) showing that after ischemic insult the protective effect of NS1619 requires expression of Kcnma1 gene. Likewise, mitochondrial ROS production stimulating complex I with 3 mM glutamate/malate was reduced by NS1619 preconditioning in the wt mice from 159+13 to 113+7 pmoles/min/mg but not in the Kcnma1-/- mice (n=3). Our results indicate that Kcnma1 gene expression is required for the improvement of mitochondrial function by NS1619 preconditioning, which involves: 1) increased CRC, and 2) decreased ROS production of complex I, both events preventing mPTP opening. Further, the results also confirm that mitoBKCa is encoded by Kcnma1 gene. Supported by NIH and AHA.