Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP]|_M) and the rate of cytosolic Ca²⁺ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca²⁺ indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial [Ca²⁺] ([Ca²⁺]_M) and, consequently, the Ca²⁺-stimulated increase in [ATP]_M, thus allowing studies of how the increase in [ATP]|_|M| and the cytosolic Ca²⁺ removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose-dependently decreased the Bk-induced increase in [Ca²⁺]_M and [ATP]_M by maximally 80% and 90%, respectively. Digital imaging microscopy of cells co-loaded with the cytosolic Ca²⁺ indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca²⁺ removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca²⁺ removal during stimulation with a Ca²⁺ mobilizing agonist. In contrast, complex I deficient patient fibroblasts displayed a cytosolic Ca²⁺ removal rate that was already decreased by 40% as compared to healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca²⁺ pumps. This work reveals the usefulness of rhodamine-based Ca²⁺ indicators in examining the role of intramitochondrial Ca²⁺ in mitochondrial (patho)physiology.