2-(4'-Pyridyl)benzimidazole (4PBI) has been used to investigate the microheterogeneity of water nanochannels of Nafion membranes at two different hydration levels. Native as well as cation exchanged Nafion membranes are found to protonate one of the two monoprotonated forms of 4PBI selectively. In native membranes and in those in which H3O+ ions are replaced by (CH3)(4)N+ ions, the pyridyl nitrogen is protonated preferentially. In Na+-exchanged membranes, however, the benzimidazole nitrogen is protonated selectively. Unlike other fluorescent probes used in earlier studies, 4PBI can differentiate between the two different cation-exchanged membranes at lower as well as higher hydration levels.