Phototropin is a flavin mononucleotide (FMN) containing blue-light receptor, which regulates, governed by its two LOV domains, the phototropic response of higher plants. Upon photoexcitation , the FMN cofactor triplet state, F-3, reacts with a nearby cysfeine to form a covalent adduct. Cysteine-to-alanine mutants of LOV domaina instead generate a flavin radical upon illumination. Here, we explore the formation of photochemically induced dynamic nuclear polarization (CISNP) in LOV2-C450A of Avena sativa phototropin and demonstrate that photo-CIDNP observed in solution C-13 NMR spectra can reliably be interpreted in terms of solid-state mechanisms including a novel triplet mechanism. To minimize cross-polarization, which transfers light-induced magnetization to adjacent C-13 nuclei, our experiments were performed on proteinjs reconstituted with specifically C-13-labeled flavins. Two potential sources for photo-CIDNP can be identified. The photogenerated triplet state. F-3, and the triplet radical pair (3)(F-center dot W+center dot), formed by electron abstraction of F-3 from tryptophan W491. To seperate the two contributions, photo-CIDNP studies were performed at four different magnetic fields ranging from 4.7 to 11.8 T. Analysis revealed that, at fields <9 T, both 3(F-center dot W+center dot) and F-3 contribute to photo-CIDNP, whereas at high magnetic fields, the calculated enhancement factors of F-3 agree favorably with their experimental counterparts. Thus, we have for the first time detected that a triplet state is the manor source for photo-CIDNP in a photoactive protein. Since triplet state are frequently encountered upon photoexcitation of flavoproteins, the novel triplet mechanism opens up new means of studying electronic structure of the active cofactors in these proteins at atomic resolution.