In time-resolved fluorescence spectroscopy, the resoln. of fluorescence species becomes increasingly difficult as their resp. lifetimes get closer. For a biexponential decay, a factor of 1.4 between the 2 decay times is commonly accepted as the practical resoln. limit. The goal of the present contribution is to characterize the fluorescence probe 5-carboxyfluorescein using frequency-domain time-resolved fluorescence spectroscopy (FD-TRFS). To resolve the different prototropic forms of this probe, the limit above had to be overcome. For this purpose, the std. global anal. method was used, and special emphasis was put on the errors assocd. with the recovered parameters. In particular, a Monte Carlo simulation was performed to est. these errors and the results of this anal. were compared with those delivered by software packages widely used in the field. The lifetimes of the trianionic and dianionic forms of 5-carboxyfluorescein were 4.01 +- 0.06 and 3.03 +- 0.09 ns, resp., and the pKa for this acid-base equil. is 6.9 +- 0.3. [on SciFinder (R)]