The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k(01) = 1.2 x 10(3) s(-1), k(21) = 1.0 x 10(11) M(-1) s(-1), k(12) = 5.5 x 10(8) S-1, k(12) = 2.2 X 10(7) S-1,and with EGTA as Ca2+ buffer: k(01) = 1.4 x 10(9) s(-1), k(12) = 5.0 x 10(10) M(-1) s(-1), k(12) = 5.5 x 10(8) S-1, k(12) = 3.2 X 10(7) S-1. k(01) and k(02) denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k(21), represents the second-order rate constant of binding of Ca2+ and Fura-e in the excited state, whereas k,, is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k(12) and k(21), the dissociation constant k(d)* in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK(d)* (3.2) is smaller than pK(d) (6.9) and that there is negligible interference of the excited-state reaction with the determination of K-d and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.