The spectroscopic properties of 2-(2'-tosylaminophenyl)benzimidazole (TPBI) have been studied in a series of different solvents. As revealed by absorbance, steady-state, and time-resolved emission spectroscopy, the molecule undergoes fast excited-state intramolecular proton transfer (ESIPT) to yield emission of the corresponding tautomeric species with a large quantum yield (0.5). The fluorescence emission shows monoexponential decay kinetics (tau = 4.5 ns) regardless of the nature of the solvent. The ground-state equilibrium is dominated by a single rotamer and small amounts of the deprotonated anion. The X-ray structure of TPBI shows a substantial out-of-plane twist along the aryl-benzimidazole bond axis, which is presumably due to T-stacking interactions between the tosylamide and benzimidazole rings. Ab initio calculations suggest a different structure in the gas phase without,pi-stacking interactions and a substantially reduced twist angle. A large energy barrier for interconversion of the cis- and trans-rotamers in the ground and excited state has been predicted on the basis of DFT calculations, which is in agreement with all experimental data. The ground-state equilibrium and ESIPT process of TPBI are essentially unaffected by the nature of the solvent, which is of particular interest to sensing applications in cell biology.