In recent years, mitochondria have emerged as important targets of agonist-dependent increases in cytosolic Ca²⁺ concentration. Here, we analyzed the significance of Ca²⁺ signals for the modulation of organelle function by directly measuring mitochondrial and cytosolic ATP levels ([ATP]_m and [ATP]_c, respectively) with specifically targeted chimeras of the ATP-dependent photoprotein luciferase. In both HeLa cells and primary cultures of skeletal myotubes, stimulation with agonists evoking cytosolic and mitochondrial Ca²⁺ signals caused increases in [ATP]_m and [ATP]_c that depended on two parameters: (i) the amplitude of the Ca²⁺ rise in the mitochondrial matrix, and (ii) the availability of mitochondrial substrates. Moreover, the Ca²⁺ elevation induced a long-lasting priming that persisted long after agonist washout and caused a major increase in [ATP]_m upon addition of oxidative substrates. These results demonstrate a direct role of mitochondrial Ca²⁺ in driving ATP production and unravel a form of cellular memory that allows a prolonged metabolic activation in stimulated cells.