Photophysics of 2-(6′-hydroxy-3′-pyridyl)benzimidazole (6-H3PyBI) has been studied with the help of absorption, fluorescence excitation and fluorescence spectroscopy, as well as, using time dependent spectrofluorimetry. Electronic structure calculations on various isomers of neutral 6-H3PyBI and different ionic species were carried out using AM1 semi-empirical calculations and density functional theory (DFT) B3LYP with 6-31G** basis set and using Gaussian 98 program. Transition absorption energies for each species have been calculated using CNDO/S-CI method, single point calculations using AM1 method and time-dependent (TD) DFT B3LYP with 6-31G** basis set. These results have confirmed the presence of three isomers/rotamers of neutral 6-H3PyBI (enol, keto-1 and keto-2), two kinds of monocations (MCE-1 and MCK-1), one kind of dication, monoanion and dianion each in S0 and S1 states. pK a and pKa* for the various equilibriums have been determined using absorption and fluorescence data and discussed. © 2004 Elsevier B.V. All rights reserved.