—Pharmacological openers of mitochondrial ATP-dependent K⁺ (mitoK_ATP) channels mimic ischemic preconditioning, and such cardioprotection can be prevented by mitoK_ATP channel blockers. It is also known that protein kinase C (PKC) plays a key role in the induction and maintenance of preconditioning. To look for possible mechanistic links between these 2 sets of observations, we measured mitochondrial matrix redox potential as an index of mitoK_ATP channel activity in rabbit ventricular myocytes. The mitoK_ATP channel opener diazoxide (100 μmol/L) partially oxidized the matrix redox potential. Exposure to phorbol 12-myristate 13-acetate (PMA, 100 nmol/L) potentiated and accelerated the effect of diazoxide. These effects of PMA were blocked by the mitoK_ATP channel blocker 5-hydroxydecanoate, which we verified to be a selective blocker of the mitoK_ATP channel in simultaneous recordings of membrane current and flavoprotein fluorescence. The inactive control compound 4α-phorbol (100 nmol/L) did not alter the effects of diazoxide. We conclude that the activity of mitoK_ATP channels can be regulated by PKC in intact heart cells. Potentiation of mitoK_ATP channel opening by PKC provides a direct mechanistic link between the signal transduction of ischemic preconditioning and pharmacological cardioprotection targeted at ATP-dependent K⁺ channels.