The photophysical behavior of a new fluorescein derivative has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength, as well as total buffer concentration, on both the absorbance and fluorescence has been investigated. The apparent acidity constant of the dye determined by absorbance is almost independent of the added buffer and salt concentrations. A semiempirical model is proposed to rationalize the variations in the apparent pK(a) values. The excited-state proton-exchange reaction around the physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the steady-state fluorescence and decay times. Fluorescence decay traces, collected as a function of total buffer concentration and pH, were analyzed by global compartmental analysis, yielding the following values of the rate constants describing excited-state dynamics: k(01) = 1.29 x 10(10) s(-1), k(02) = 4.21 x 10(8) s(-1), k(21)approximate to 3 x 10(6) M-1 s(-1), k(12)(B) = 6.40 x 10(8) M-1 s(-1), and k(21)(B) = 2.61 x 10(7) M-1 s(- 1). The decay rate constant values of k(01), k(21), and k(21)(B), along with the low molar absorption coefficient of the neutral form, mean that coupled decays are practically monoexponential at buffer concentrations higher than 0.02 M and any pH. Thus, the pH and buffer concentration can modulate the main lifetime of the dye.