Flavoprotein radicals are important intermediates in many biochemical processes. In the blue light sensor plant cryptochrome, the radical state acts as a signaling state. An isolation and assignment of infrared bands of flavin radicals in the most relevant spectral region of carbonyl stretches is missing because of their overlap with absorption of water and the protein moiety. In this study, the neutral radical state of flavoproteins was investigated by Fourier transform infrared difference spectroscopy. The light-induced conversion of oxidized to neutral radical state was monitored in a plant cryptochrome and that of radical to fully reduced state in a DASH cryptochrome. A pure difference spectrum of flavin radical minus oxidized state was obtained from a point mutant of a phototropin LOV (light-, oxygen-, or voltage-sensitive) domain. The analysis of the spectra revealed a correlation between the frequencies of carbonyl vibrations of the flavin radical state and those of its visible absorption. Plant cryptochrome shows a very low frequency of the carbonyl stretch in the radical state. It is postulated that the downshift is caused by the charge of an adjacent aspartate, which donated its proton to flavin N-5. Contributions from the protein moiety to the spectra were isolated for DASH and plant cryptochromes. As a conclusion, the photosensitive domain of plant cryptochromes shows changes in secondary structure upon illumination, which might be related to signaling.