Significance Mitochondrial functions depend on the steep H ⁺ electrochemical gradient (ΔμH ⁺ ) across their inner membrane. The available tools for controlling this gradient are essentially limited to inhibitors of the respiratory chain or of the H ⁺ ATPase or to uncouplers, poisons plagued by important side effects and that lack both cell and spatial specificity. We show here that, by transfecting cells with the cDNA encoding channelrhodopsins specifically targeted to the inner mitochondrial membrane, we can obtain an accurate and spatially confined, light-dependent control of mitochondrial membrane potential and, as a consequence, of a series of mitochondrial activities ranging from electron transport to ATP synthesis and Ca ²⁺ signaling.