Muscle contraction requires ATP and Ca²⁺, and thus, is under direct control of mitochondria and the sarcoplasmic reticulum. Since aerobic ATP production is enhanced by Ca²⁺ stimulation of mitochondrial dehydrogenases and the ATP synthase, we characterized mitochondrial Ca²⁺ uptake and superoxide flash (mSOF) production, stochastic events of quantal mitochondrial superoxide generation, in single mouse flexor digitorum brevis (FDB) muscle fibers. Repetitive stimulation (500ms, 100Hz, every 2.5s) triggered a remarkably sustained mitochondrial Ca²⁺ uptake after both 5 (peak Rhod-2 fluorescence increased 412±45%) and 40 tetani (662 ±55%), which decayed only slowly over the subsequent 10 minutes. Unexpectedly, the same stimulation protocol regulated mSOF activity in a biphasic manner. Specifically, mSOF frequency was significantly increased (27±8.9%) following 5 tetani and markedly decreased (56±6.6%) after 40 tetani. In addition, mSOF activity depended strongly on electron transport chain (ETC) and adenine nucleotide translocase functionality, but was independent of cyclophilin-D-mediated mitochondrial permeability transition pore activity. Diverse spatial dimensions of individual mSOF events reflected a complex underlying morphology of the mitochondrial network, as examined by electron microscopy. These results demonstrate that mitochondrial Ca²⁺ uptake and ETC activity in skeletal muscle are tightly coupled to activity-dependent changes in the cellular metabolic state.