We here present a novel method, based on the targeting of the photoprotein aequorin, for measuring the concentration of Ca2+ ions in defined cellular compartments of intact cells. In this contribution we will discuss the application to mitochondria. A chimaeric cDNA was constructed by fusing in frame the aequorin cDNA with that for a mitochondrial protein. The cDNA encoded a "mitochondrially-targeted" aequorin, composed of a typical mitochondrial targeting signal at the N-terminus and the photoprotein at the C-terminus. The cDNA, inserted in the expression vector pMT2, was co-transfected into bovine endothelial and HeLa cells together with the selectable plasmid pSV2-neo and stable transfectants, selected for high aequorin production, were analyzed. In subcellular fractionations, aequorin was shown to be localized in mitochondria; in intact cells, the first direct measurement of mitochondrial free Ca2+, [Ca2+]m, were obtained, which showed that [Ca2+]m is low at rest (< 0.5 microM), but rapidly increases to the micromolar range upon cell stimulation [1]. These data indicate that mitochondria "sense" very accurately the cytosolic Ca2+ concentration ([Ca2+]i), and after cell stimulation [Ca2+]m rises to values capable of activating the Ca(2+)-sensitive mitochondrial dehydrogenases.