Mitochondria sense cytoplasmic Ca2+ signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca2+ signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca2+ signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca2+ signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca2+ transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca2+ signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca2+ signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45Hz) skeletal myotubes. Similar Ca2+ signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca2+ rise induced by stimulation was inhibi