In isolated, catalytically inactive, DNA photolyase from Escherichia coli (E. coli), the flavin adenine dinucleotide cofactor is in its neutral radical state FADH(.). It can be activated by a unique light-induced reduction of the flavin, a process initiated by the formation of the excited-state FADH(.)*. As the photophysical properties of this state are essentially unknown, we performed a comparative characterization by femtosecond transient absorption spectroscopy of FADH(.)* in DNA photolyase from E. coli and glucose oxidase from Aspergillus niger, and of the excited neutral radical flavin mononucleotide (FMNH.*) in flavodoxin from Desulfovibrio gigas. In contrast to photolyase, in glucose oxidase and flavodoxin no electron-transfer products are observed after selective excitation of the flavin radical. In glucose oxidase, FADH(.)* decays to the ground state in 59 +/- 15 ps, close to the 80-ps intrinsic lifetime of the excited state in photolyase, and we discuss that the intrinsic lifetime of the excited state of flavin radical in protein environment is in the 50-80 ps range. FMNH.* in flavodoxin decays much faster (2.3 +/- 0.3 ps), possibly because of quenching by formation of a very short-lived (< 0.7 ps) electron-transfer intermediate. Spectroscopically, the excited state of FADH* in photolyase displays a pronounced spectral feature that is absent in the other systems studied. Further characterization by polarized photoselection experiments identifies the feature as an additional induced absorption band at similar to 550 nm superimposed on the ground-state bleaching signal. In view of the unique U-shape configuration of FAD in photolyase, we suggest it to reflect a flavin-adenine charge-transfer interaction.