Fluorescence properties of fluorescein-based probes are shown to be finely controlled by the rate of photoinduced electron transfer from the benzoic acid moiety (electron donor) to the singlet excited state of the xanthene moiety (electron acceptor fluorophore). The occurrence of photoincluced electron transfer is clearly evidenced by transient absorption spectra showing bands due to the radical cation of the electron donor moiety and the radical anion of the xanthene moiety, observed in laser flash photolysis experiments. The photoinduced electron transfer rates and the rates of back electron transfer follow the Marcus parabolic dependence of electron transfer rate on the driving force. Such a dependence provides for the first time a quantitative basis for a rational design principle which has high efficiency in modulating fluorescence properties of fluorescein-based probes.