We have expressed aequorin in mitochondria of the yeast Saccharomyces cerevisiae and characterized the resulting strain with respect to mitochondrial Ca 2+ transport in vivo and in vitro. When intact cells are suspended in water containing 1.4 mM ethanol and 14 mM CaCl 2, the matrix free Ca 2+ concentration is 200 nM, similar to the values expected in cytoplasm. Addition of ionophore ETH 129 allows an active accumulation of Ca 2+ and promptly increases the value to 1.2 μM. Elevated Ca 2+ concentrations are maintained for periods of 6 min or longer under these conditions. Isolated yeast mitochondria oxidizing ethanol also accumulate Ca 2+ when ETH 129 is present, but the cation is not retained depending on the medium conditions. This finding confirms the presence of a Ca 2+ release mechanism that requires free fatty acids as previously described [P.C. Bradshaw et al. (2001) J. Biol. Chem. 276, 40502–40509]. When a respiratory substrate is not present, Ca 2+ enters and leaves yeast mitochondria slowly, at a specific activity near 0.2 nmol/min/mg protein. Transport under these conditions equilibrates the internal and external concentrations of Ca 2+ and is not affected by ruthenium red, uncouplers, or ionophores that perturb transmembrane gradients of charge and pH. This activity displays sigmoid kinetics and a K 1/2 value for Ca 2+ that is near to 900 nM, in the absence of ethanol or when it is present. It is furthermore shown that the activity coefficient of Ca 2+ in yeast mitochondria is a function of the matrix Ca 2+ content and is substantially larger than that in mammalian mitochondria. Characteristics of the aequorin-expressing strain appear suitable for its use in expression-based methods directed at cloning Ca 2+ transporters from mammalian mitochondria and for further examining the interrelationships between mitochondrial and cytoplasmic Ca 2+ in yeast.