We examined the effects of divalent cations on the fluorescence excitation spectra of fura-2 from a 2 ml solution containing 1 microM fura-2 with an excitation wavelength of 300-425 nm and an emission wavelength of 500 nm. The fluorescence spectra in solutions of fura-2-Ca2+, fura-2-Ba2+, and fura-2-Sr2+ complexes show almost the same pattern. The dissociation constants (Kd) of Ba2+ and Sr2+ with fura-2 were 1.63 and 9.02 microM, respectively, and these values were higher than that of the Ca2+ complex (222 nM). The effect of Mg2+ binding on the fluorescence of fura-2 was quite small, and Mn2+ quenched the fluorescence of fura-2. The spectra of the fura-2-Ca2+ complex were changed by the co-existence of another divalent cation. Divalent cations competitively bind to the binding site of fura-2 and show the respective fluorescence spectra; consequently, the composed fluorescence spectra according to the respective Kd.