Upon physiological stimulation, mitochondria undergo a major rise in mitochondrial [Ca2+] ([Ca2+]m) in a wide variety of cell types. Here, particular attention will be focused on the mechanism that allows the low-affinity transporters of mitochondria to rapidly accumulate Ca2+, despite the low amplitude of the cytosolic [Ca2+] ([Ca2+]c) rises, i.e. the close apposition of mitochondria to the Endoplasmic Reticulum (ER), the main pool of agonist-releasable Ca2+. Upon opening of IP3-gated channels, mitochondria are able to sense not the average [Ca2+]c rise, but rather the much higher concentration occurring in the proximity of the open channels. We will then address the functional significance of this process, that spans from the activation of organelle metabolism to the alteration of organelle morphology, and consequent release of pro-apoptotic factors during apoptosis.