Description: In skeletal muscle, resting intracellular Ca 2+ concentration ([Ca 2+ ] i ) homeostasis is exquisitely regulated by Ca 2+ transport across the sarcolemmal, mitochondrial, and sarcoplasmic reticulum (SR) membranes. Of these three systems, the relative importance of the mitochondria in [Ca 2+ ] i regulation remains poorly understood in in vivo skeletal muscle. We tested the hypothesis that the capacity for Ca 2+ uptake by mitochondria is a primary factor in determining [Ca 2+ ] i regulation in muscle at rest and following contractions. Tibialis anterior muscle of anesthetized peroxisome proliferator-activated receptor- coactivator-1α (PGC-1α)-overexpressing (OE, increased mitochondria model) and wild-type (WT) littermate mice was exteriorized in vivo and loaded with the fluorescent probe fura 2-AM, and Rhod 2-AM Ca 2+ buffering and mitochondrial [Ca 2+ ] were evaluated at rest and during recovery from fatiguing tetanic contractions induced by electrical stimulation (120 s, 100 Hz). In addition, the effects of pharmacological inhibition of SR (thapsigargin) and mitochondrial [carbonyl cyanide- 4 -(trifluoromethoxy) phenylhydrazone (FCCP)] function were examined at rest. [Ca 2+ ] i in WT remained elevated for the entire postcontraction recovery period (+6 ± 1% at 450 s), but in PGC-1α OE [Ca 2+ ] i returned to resting baseline within 150 s. Thapsigargin immediately and substantially increased resting [Ca 2+ ] i in WT, whereas in PGC-1α OE this effect was delayed and markedly diminished (WT, +12 ± 3; PGC-1α OE, +1 ± 2% at 600 s after thapsigargin treatment, P 〈 0.05). FCCP abolished this improvement of [Ca 2+ ] i regulation in PGC-1α OE. Mitochondrial [Ca 2+ ] accumulation was observed in PGC-1α OE following contractions and thapsigargin treatment. In the SR, PGC-1α OE downregulated SR Ca 2+ -ATPase 1 (Ca 2+ uptake) and parvalbumin (Ca 2+ buffering) protein levels, whereas mitochondrial Ca 2+ uptake-related proteins (Mfn1, Mfn2, and mitochondrial Ca 2+ uniporter) were upregulated. These data demonstrate a heretofore unappreciated role for skeletal muscle mitochondria in [Ca 2+ ] i regulation in vivo following fatiguing tetanic contractions and at rest.