On the basis of extraction experiments, the exchange extraction constant corresponding to the equilibrium H3O+(aq) + 1.Na+(nb) 1.H3O+(nb) + Na+(aq) occurring in the two–phase water–nitrobenzene system (1 = enniatin B; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log Kex (H3O+, 1.Na+) = 0.9 ± 0.1. Further, the stability constant of the 1.H3O+ complex in nitrobenzene saturated with water was calculated for a temperature of 25 oC: log βnb (1.H3O+) = 6.4 ± 0.2. Finally, by using quantum mechanical DFT calculations, the most probable structure of the cationic complex species 1.H3O+ was derived. In the resulting complex, the “central” cation H3O+ is bound by three relatively strong hydrogen bonds to the corresponding three carbonyl oxygens of the parent enniatin B ligand. The interaction energy, E(int), of the considered complex 1.H3O+ was found to be -424.8 kJ/mol, confirming also the formation of this investigated complex.