The photophysical behavior of BCECF [2', 7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein]-currently the most widely used fluorescent pH indicator for near-neutral intracellular pH measurements-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 the absorbance and steady-state fluorescence has been investigated. The apparent acidity constant of the pH indicator determined by absorbance and fluorescence titration is dependent on 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 of BCECF at physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the fluorescence 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 of BCECF: k(01) = 3.4 x 10(8) s(-1), k(02) = 2.6 x 10(8) s(-1), k(21) approximate to 1 x 10(6) M-1 s(-1), k(12)(B) = 1.4 x 10(8) M-1 s(-1), and k(21)(B) = 4.3 x 10(7) M-1 s(-1).