Valinomycin, a cyclic peptide, was incorporated into a biomimetic lipid membrane tethered to' the surface of a gold (111) electrode. Electrochemical impedance spectroscopy was used to study the ionophore properties of the peptide, and polarization modulation infrared reflection absorption spectroscopy was employed to determine the conformation and orientation of valinomycin in the membrane. The combination of these two techniques provided unique information about the ionophore mechanism where valinomycin transports ions across the membrane by creating a complex with potassium ions and forming an ion pair with a counter anion. The ion pair resides within the hydrophobic fragment of the membrane and adopts a small angle of similar to 22 degrees with respect to the surface normal. This novel study provides new insights explaining the valinomycin ion transport mechanism in model biological membranes.