Time-resolved polarized fluorescence experiments have been carried out on the FAD of tetrameric NADH peroxidase from Enterococcus faecalis and three mutant enzymes, C42A, C42S, and Y159A, respectively. In particular Tyr159 and, in part, Cys42 turned out to be the amino acids which are responsible for the strong dynamic quenching of flavin fluorescence, because two picosecond fluorescence lifetime components <150 ps are clearly present in the wild-type enzyme and in the Cys42 mutants, while only one picosecond lifetime <150 ps is present in the Tyr159 mutant. This observation is corroborated by the distance information obtainable from the known three-dimensional structure of the wild-type enzyme. Steady-state fluorescence spectroscopy indicated that the Tyr159 mutant has the same fluorescence yield as both Cys42 mutants suggesting that static fluorescence quenching prevails in the tyrosine mutant. Cys42 is the amino acid which is probably responsible for the static quenching in the wild-type enzyme and Y159A mutant. The time-resolved fluorescence anisotropy data showed a dependence on the emission wavelength. In case of proteins with Tyr159 present, less rapid depolarization is observed when the emission wavelength is at 526 nm, while depolarization of a few nanoseconds is more clearly visible at 568 nm. The rapid depolarization process was absent in the Y159A mutant irrespective of emission wavelength. The latter protein only showed a minor component of relatively long correlation time (>10 ns) which can be attributed to energy transfer among the flavins in the tetramer. The rapid ns depolarization is due to excited-state charge transfer between Tyr159 and flavin, which leads to a change of transition moment out of the plane of the isoalloxazine ring. The latter process contributes to a major extent to the observed fluorescence anisotropy decay and can be considered as an unusual source of fluorescence depolarization.