The transfer of Ca(2+) from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca(2+) ([Ca(2+)](cyto)) can be excellently quantified with the ratiometric Ca(2+) probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca(2+) sensors, the cameleons, are efficiently used to specifically measure Ca(2+) within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca(2+)](cyto) and mitochondrial Ca(2+) ([Ca(2+)](mito)) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca(2+) probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca(2+)](cyto) and [Ca(2+)](mito) within same individual cells. Our data indicate that depending on the kinetics of [Ca(2+)](cyto) rises there is a significant lag between onset of [Ca(2+)](cyto) and [Ca(2+)](mito) signals, pointing to a certain threshold of [Ca(2+)](cyto) necessary to activate mitochondrial Ca(2+) uptake. The temporal correlation between [Ca(2+)](mito) and [Ca(2+)](cyto) as well as the efficiency of the transfer of Ca(2+) from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca(2+) extrusion and a desensitization of mitochondrial Ca(2+) uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca(2+) oscillations of pancreatic beta-cells in response to D-glucose.