Mitochondria promptly respond to Ca(2+)-mediated cell stimulations with a rapid accumulation of the cation into the matrix. In this article, we review (i) the basic principles of mitochondrial Ca(2+) transport, (ii) the physiological/pathological role of mitochondrial Ca(2+) uptake, (iii) the regulatory mechanisms that may operate in vivo, and (iv) the new targeted Ca(2+) probes that allowed the "rediscovery" of these organelles in calcium signalling.