We have recently developed a new method for monitoring Ca2+ concentrations in defined cell compartments. The cDNA encoding the Ca(2+)-sensitive photoprotein aequorin has been modified in order to include specific targeting sequences and expressed in eukaryotic cells; the recombinant protein, specifically located inside the cells, has allowed the direct study of mitochondrial and nuclear Ca2+ concentrations in living cells. The principles, and the application, of this new methodology are discussed in this article.