Fluo-3, a fluorescent Ca2+ indicator, is sequestered by isolated rat liver mitochondria and is an effective probe for evaluating the concentration and kinetics of change of mitochondrial matrix ionized calcium ([Ca2+]m) under a variety of conditions. At the wavelengths employed, there is no significant interference by auto-fluorescence. There is an insignificant release of the indicator over four hours and the loading and presence of fluo-3 has no effect on respiratory rate or oxidative phosphorylation. The [Ca2+]m steady state can be altered by the assay conditions, i.e. the presence of extra-mitochondrial Ca2+, Mg2+ phosphate and respiratory inhibitors. The total matrix ionized calcium represents a small percent (less than 0.01%) of the total mitochondrial calcium.