Massive amounts of Ca2+ can accumulate in mitochondria, owing to its complexation with matrix phosphate. Under conditions in which the mito-chondrial uniporter is the foremost pathway for Ca 2+ efflux, the release of sequestered Ca 2+ by protonophoric uncouplers is invariably demonstrated. This has been recently ascribed to matrix acidification, which results in the dissociation of the Ca 2+ –phosphate complex.