Excited state intramolecular proton transfer (ESIPT) has been studied in 2-(2′-amino-3-pyridyl)benzimidazole (2-A3PyBI) in different solvents with the help of absorption, fluorescence excitation and fluorescence spectroscopy and time resolved emission spectrofluorimetry. Electronic structure calculations on each species were carried out using semi-empirical AM1 method and hybrid density functional theory (DFT) B3LYP using 6-31 G** basis set and Gaussian 98 program. Dual fluorescence is observed in all the solvents. Based on theoretical and experimental results, normal small Stokes shifted emission is assigned to rotamer a-2 in non-polar solvents and a-3 in polar protic solvents. Long wavelength (LW) and large Stokes shifted band is due to tautomer formed by ESIPT from rotamer a-1. Presence of electron withdrawing =N-atom ortho to -NH 2 group increases its acidity. Due to this rate of ESIPT process will be enhanced and thus the fluorescence quantum yield (Φ f T) of the tautomer will also be increased. Both AM1 and DFT calculations have shown that a-1 is the most stable among all the rotamers and semi-empirical calculations have indicated that the activation barrier for interconvertion of a-1 to a-2 is only 2.6 kJ mol -1 in the ground state (S 0) and 44.3 kJ mol -1 in the first excited singlet (S 1). On the other hand, activation barrier height for the conversion of a-1 to a-T is 132 kJ mol -1 in S 0 state and 72 kJ mol -1 in S 1 state, obtained by AM1 method. © 2004 Elsevier B.V. All rights reserved.