Ischemic preconditioning, or the protective effect of short ischemic episodes on a longer, potentially injurious, ischemic period, is prevented by antagonists of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) and involves changes in mitochondrial energy metabolism and reactive oxygen release after ischemia. However, the effects of ischemic preconditioning itself on mitochondria are still poorly understood. We determined the effects of ischemic preconditioning on isolated heart mitochondria and found that two brief (5 min) ischemic episodes are sufficient to induce a small but significant decrease (∼25%) in mitochondrial NADH-supported respiration. Preconditioning also increased mitochondrial H₂O₂ release, an effect related to respiratory inhibition, because it is not observed in the presence of succinate plus rotenone and can be mimicked by chemically inhibiting complex I in the presence of NADH-linked substrates. In addition, preconditioned mitochondria presented more substantial ATP-sensitive K⁺ transport, indicative of higher mitoK_ATP activity. Thus we directly demonstrate that preconditioning leads to mitochondrial respiratory inhibition in the presence of NADH-linked substrates, increased reactive oxygen release, and activation of mitoK_ATP.