Discovered in the cardiac sarcolemma, ATP-sensitive K⁺(K_ATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial K_ATP channel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K⁺ channel openers to examine the effect of mitochondrial K_ATPchannel opening on mitochondrial membrane potential, respiration, ATP generation, Ca²⁺ transport, and matrix volume. From a mitochondrial membrane potential of −180 ± 15 mV, K⁺ channel openers, pinacidil (100 μM), cromakalim (25 μM), and levcromakalim (20 μM), induced membrane depolarization by 10 ± 7, 25 ± 9, and 24 ± 10 mV, respectively. This effect was abolished by removal of extramitochondrial K⁺ or application of a K_ATP channel blocker. K⁺ channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K⁺ channel opener released Ca²⁺ from mitochondria preloaded with Ca²⁺, an effect also dependent on extramitochondrial K⁺concentration and sensitive to K_ATP channel blockade. In addition, K⁺ channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome cand adenylate kinase. Thus, in isolated cardiac mitochondria, K_ATP channel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca²⁺, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial K_ATP channels in regulating functions vital for the cardiac mitochondria.