<p>Cells cope with salt stress, hypoxia or elevated cytosolic Ca²⁺ by regulating their mitochondrial Na⁺ levels. The discovery of the mitochondrial Na⁺/Ca²⁺ exchanger and its disease relevance has revealed the need to map mitochondrial Na⁺ in situ. Here we describe a ratiometric fluorescent reporter for Na⁺, denoted MitRatiNa, that reports mitochondrial Na⁺ levels independent of membrane potential and in diverse cell lines. Na⁺ in individual mitochondria varies greatly and, depending on cell type, can be as low as 1–5 mM or as high as 40 mM on average. We demonstrate that mitochondrial Na⁺ increases during cytosolic Ca²⁺ elevation, inhibition of glycolysis or respiration. Mitochondria in skin fibroblasts from healthy humans show a high Na⁺ population that disappears in fibroblasts of persons with mitochondrial diseases. The newfound ability to map absolute Na⁺ at the resolution of single mitochondria enables the dissection of regulatory mechanisms for mitochondrial Ca²⁺ and Na⁺ and potential identification of new therapeutic avenues.</p><p></p>