YtvA from Bacillus subtilis was found as the first prokaryotic phototropin-like blue-light-responsive photoreceptor. It is composed of two domains, the photoactive LOV (light, oxygen, voltage) domain, which binds a flavin mononucleotide (FMN) as a chromophore and a STAS (sulfate transporter/anti-sigma-factor antagonist) domain, which generates a physiological signal. Here we present a routine chromophore-exchange protocol that allows chemically synthesized, structurally modified chromophores instead of the naturally present flavin mononucleotide (FMN) chromophore to be introduced. FMN was exchanged for riboflavin (RF), flavin adenine dinucleotide (FAD), 7,8-didemethyl flavin mononucleotide (DMFMN), and 8-isopropyl flavin mononucleotide (iprFMN). LOV domains reconstituted with new flavins undergo the same photocycle as native YtvA LOV, consisting of triplet formation and covalent binding of the chromophore followed by a thermal recovery of the parent state, albeit with different kinetics and photophysical properties. Interestingly, the iprFMN chromophore, inducing steric hindrances to the protein, exhibits a very fast light-to-dark-conversion and shows a high fluorescence quantum yield (0.4). Incorporation of FAD causes an increase of its fluorescence quantum yield from 0.04 (H 2O) to 0.2. Shuffling chromophores: A successful, routine chromophore-exchange protocol allows chemically synthesized, structurally modified chromophores (used chromophores see on the formula) instead of the naturally present flavin mononucleotide (FMN) chromophore to be introduced under mild, protein-friendly conditions. The photochemical properties of newly incorporated flavin derivatives in YtvA's LOV domain were investigated and described. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.