Mitochondrial ability of shaping Ca(2+) signals has been demonstrated in a large number of cell types, but it is still debated in heart cells. Here, we take advantage of the molecular identification of the mitochondrial Ca(2+) uniporter (MCU) and of unique targeted Ca(2+) probes to directly address this issue. We demonstrate that, during spontaneous Ca(2+) pacing, Ca(2+) peaks on the outer mitochondrial membrane (OMM) are much greater than in the cytoplasm because of a large number of Ca(2+) hot spots generated on the OMM surface. Cytoplasmic Ca(2+) peaks are reduced or enhanced by MCU overexpression and siRNA silencing, respectively; the opposite occurs within the mitochondrial matrix. Accordingly, the extent of contraction is reduced by overexpression of MCU and augmented by its down-regulation. Modulation of MCU levels does not affect the ATP content of the cardiomyocytes. Thus, in neonatal cardiac myocytes, mitochondria significantly contribute to buffering the amplitude of systolic Ca(2+) rises.