The incorporation of valinomycin molecules into cadmium stearate multilayers increased complexation of valinomycin with potassium ions and potassium permeability of the multilayers. Valinomycin-cadmium stearate mixed Langmuir-Blodgett multilayers were exposed to aqueous solutions of KCl, NaCl and KCl NaCl mixtures in a concentration range of 0.001–100 mM. The current responses of the multilayers indicated a significant dependence on the K + concentration but not on the Na + concentration. A marked preference for K + over Na + was observed in mixed ion solutions even when the concentration of Na + exceeded the concentration of K + by the factor of 100000. The K + Na + selectivity was dependent on the concentration of valinomycin in mixed monolayers and was highest at about 9 mol% of valinomycin, at which point the antibiotic molecules were completely surrounded by cadmium stearate molecules. This information may be useful for understanding of fundamental properties of molecular assemblies and antibiotic/cell membrane interactions.