Although the importance of mitochondria in patho-physiology has become increasingly evident, it remains unclear whether these organelles play a role in Ca²⁺ handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring Ca²⁺ transients reliably during the contraction–relaxation cycle. Using two-photon microscopy and genetically expressed “cameleon” Ca²⁺ sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial Ca²⁺ transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up Ca²⁺ during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial Ca²⁺ increase is delayed by a few milliseconds compared with the cytosolic Ca²⁺ rise and occurs both during a single twitch and upon tetanic contraction.