A physicochemical study of the Mag-indo1 binding to Ca²⁺ in solution showed that: (i) the characteristic fluorescence spectra of Ca²⁺-bound and Mg²⁺-bound Mag-indo1 are identical; (ii) two successive equilibria occur for increasing Ca²⁺ concentrations; and (iii) the value of the dissociation constant of the first one, as determined by using a probe dilution protocol, amounts to 780 nM. In order to investigate the fluorescence level of Mag-indo1 trapped in cell organelles, fluorescence spectra of Mag-indo1-loaded fibroblasts were recorded before and after a digitonin permeabilization. Their resolution into cation-bound, protein-bound, and free Mag-indo1 characteristic spectra allowed measurement of the fluorescence intensities of these species. The intensities emitted from whole cells were compared to those emitted from organelles (assumed to be endoplasmic reticulum according to a DiOC₆ loading). The cation-bound Mag-indo1 fluorescence resulted partially (20 to 50%) from the cytosol for 30% of the cells, and totally from compartments for 70% of the cells. We found a concentration value of 500 nM for compartmentalized Ca²⁺ and concluded that the Mag-indo1 binding to Ca²⁺ is likely to affect drastically the Mg²⁺ concentration measurements in cells. Moreover, we showed that the amount variation of protein-bound Mag-indo1 also affects Mg²⁺ measurements when using the two-wavelength ratio method.