Mitochondria must maintain volume homeostasis in order to carry out oxidative phosphorylation. It has been postulated that the concentration of free Mg²⁺ ([Mg²⁺]) serves as the sensor of matrix volume and regulates a K⁺-extruding K⁺/H⁺ antiport (K. D. Garlid. J. Biol. Chem. 255: 11273-11279, 1980). To test this hypothesis, the fluorescent probe furaptra was used to monitor [Mg²⁺] and free Ca²⁺ concentration ([Ca²⁺]) in the matrix of isolated beef heart mitochondria, and K⁺/H⁺ antiport activity was measured by passive swelling in potassium acetate. Concentrations that result in 50% inhibition of maximum activity of 92 µM matrix [Mg²⁺] and 2.2 µM [Ca²⁺] were determined for the K⁺/H⁺ antiport. Untreated mitochondria average 670 µM matrix [Mg²⁺], a value that would permit <1% of maximum K⁺/H⁺ antiport activity. Hypotonic swelling results in large decreases in matrix [Mg²⁺], but swelling due to accumulation of acetate salts does not alter [Mg²⁺]. Swelling in phosphate salts decreases matrix [Mg²⁺], but not to levels that permit appreciable antiport activity. We conclude that 1) it is unlikely that matrix [Mg²⁺] serves as the mitochondrial volume sensor, 2) if K⁺/H⁺ antiport functions as a volume control transporter, it is probably regulated by factors other than [Mg²⁺], and 3) alternative mechanisms for mitochondrial volume control should be considered.