The acidity constants of 16 2-substituted 5-nitrobenzimidazole derivatives were determined in dimethyl sulfoxide, 60% (v/v) isopropyl alcohol +40% (v/v) water, and 60% (v/v) dimethyl sulfoxide +40% (v/v) water by the potentiometric method. Tetrabutylammonium hydroxide was used as the titrant for the measurements done in dimethyl sulfoxide as a solvent, and aqueous KOH was used for the measurements done in mixtures. The pK(a),values were obtained from the half-neutralization potentials which were obtained from an analysis of the potentiometric titrations. The order of the acidic strengths obtained depends on the alkyl, aryl, and heterocyclic substituents on 5-nitrobenzimidazole. The acidic strengths of the alkyl-substituted 5-nitrobenzimidazoles in each solvent were in the following order: 2-(trifluoromethyl) > 2-(difluorochloromethyl) > 2-(chloromethyl) > 3-nitrobenzimidazole > 2-methyl > 2-(hydroxymethyl)-5-nitrobenzimidazole. The acidic strengths of the aryl-substituted 5-nitrobenzimidazoles in each solvent were in the following order: 2-(4'-chlorophenyl) > 2-(2'-chlorophenyl) > 2-(3'-chlorophenyl) > 2-(2'-hydroxyphenyl) > 2-phenyl > 5-nitrobenzimidazole > 2-(4'-methoxyphenyl) > 5-nitrobenzimidazole. The increasing acidic strengths of the heterocyclic-group-substituted 5-nitrobenzimidazoles for three solvent systems were in the order 2-(2'-thienyl) > 2-(2-pyridyl) > 2-(3'-pyridyl) > 2-(2'-furyl) > 5-nitrobenzimidazole.