KGaA, Weinheim We report the creation of a novel group of the ABDI-BF2fluorescent dyes based on the conformationally locked GFP chromophore. We studied the intramolecular mechanism of radiationless deactivation of the ABDI-BF2fluorophore by introducing the various substituents at the nitrogen atom. The results of this study and our previous work allowed us to claim that in case of ABDI-BF2this deactivation is determined by the formation of the nonfluorescent internal charge transfer exited state with a planar quinoidal structure. The electronic effects have a greater impact on the radiationless deactivation than the conformation. Thus, the introduction of an electron-donating group is more effective than the creation of rigid derivatives. The presented dyes are characterized by high fluorescence quantum yields and pH-independence in the physiological pH range, making them promising candidates for a wide spectrum of fluorescence labeling applications.