It has been theoretically predicted that under conditions leading to Gibbs-Donnan equilibrium in case when size of one compartment is very different from another (as in system “membrane vesicle/liposomes—incubation medium”) stable transmembrane potential can be formed, which value is sufficient to fit requirement of real transmembrane potential. Four partial cases were considered with different location and charge of impermeable ion and it was concluded that locations of impermeable ions in medium provide stable transmembrane potential with sufficient value of 60 - 70 mV. Potential-sensitive probe, such as DiOC₆(3) and oxonol VI, were used to confirm the calculated potential. According to the change in fluorescence level and emission/excitation shift, a stable and relatively high transmembrane potential can be formed if salt of impermeable ion is located in incubation medium. Impermeable cations and anions may be used to create positive and negative transmembrane potential respectively.