Background. A large body of evidence unequivocally demonstrated that pharmacological modulation of the mitochondrial ATP-sensitive potassium channels (mitoKATP) is protective against myocardial ischemia/reperfusion injury. The present work was aimed at assessing the dose-dependent effect of four novel mitoKATP openers – benzopyranyl analogues (L-1487, KL-1492, KL-1495, and KL-1507) on mitochondrial respiration and reactive oxygen species production in isolated rat heart mitochondria. Materials and methods. Experiments were performed on Sprague Dawley (SD) adult female rats (4-6 months, n = 5-6/group) anesthetized by the intraperitoneal administration of a mixture of ketamine (30 mg/kg) and xylazine (5 mg/ kg). Rat heart mitochondria were isolated by differential centrifugations at 40C. Mitochondrial respiratory function was assessed by high-resolution respirometry at 370C (Oxygraph-2k, Oroboros Ltd.). Hydrogen peroxide (H2O2) release was determined with the Amplex Red fluorescence assay. Four different concentrations: 50, 75, 100 and 150 μM of the compounds were tested. Results. When applied in higher concentrations (100 and 150 μM) a significant increase of state 2 and state 4 respiratory rates for mitochondria respiring in the presence of both complex I (CI) and complex II (CII) substrates was found. However, the maximal concentration (150 μM) elicited an important decrease of the oxidative phosphorylation. When applied in the higher concentrations (100 and 150 μM) all investigated compounds determined a significant decrease of mitochondrial H2O2 release in mitochondria respiring on complex I substrates (glutamate/malate). Conclusion. The novel mitochondrial KATP openers are able to elicit mild uncoupling and decreased oxidative stress in isolated rat heart mitochondria, properties that are likely to be effective in cardioprotection