An excited-stare intramolecular proton transfer (ESIPT) process has been observed in the anionic species of 2,-(2'-acetamidophenyl)benzimidazole (2-AMPBI). Dual fluorescence is observed in the case of monoanionic species. The presence of different monoanionic species has been characterized by means of steady-state and time-resolved fluorescene spectroscopy, as well as using AM1 semi-empirical quantum-mechanical calculations. The present study revealed that the presence of an acetyl group plays a vital role in the stability of different ionic and neutral species and in changes in their ground (S-0) and excited singlet state (S-1) acid-base properties with respect to 2-(2'-aminophenyl)benzimidazole (2-APBI).