Exercise provides cardioprotection against ischemia-reperfusion injury, a process involving mitochondrial reactive oxygen species (ROS) and calcium overload. Adaptive strategies within mitochondria that could contribute to protection include decreased ROS production and increased tolerance to calcium overload. This study tested the hypotheses that endurance training decreases ROS production and improves tolerance against Ca²⁺-induced dysfunction in myocardial mitochondria. Male F344 rats were either sedentary (SED, n=8) or endurance-trained (ET, n=11) by running on a treadmill for 16 weeks (5-days/week, 60 min/day, 25 m/min, and 6° grade). Mitochondrial oxidative phosphorylation measures were determined with glutamate/malate or succinate as substrates and H₂O₂ production and permeability transition pore (PTP) opening determined with succinate. As CaCl₂ increased from 0 to 50 µM, SED and ET displayed similar progressive decreases in State 3 respiration, respiratory control ratio, and ADP:O ratio. Ca²⁺-induced PTP opening was also similar. However, H₂O₂ production by ET was lower than SED (P<0.05) in the absence of calcium (323±12 vs 362±11 pmol/min/mg protein) and the presence of 50 µM CaCl₂ (154±3 vs 197±7). Rotenone, which blocks electron flow from succinate to complex 1, reduced H₂O₂ production and eliminated differences between ET and SED. Mitochondrial superoxide dismutase and glutathione peroxidase were not affected by exercise. Catalase activity was extremely low, but increased 49% in ET (P<0.05). We conclude that exercise reduces ROS production in myocardial mitochondria through adaptations specific to complex 1, but does not improve mitochondrial tolerance to calcium overload.