Self-assembled lipid layers (SALL) have been formed on a platinum electrode surface from aqueous solutions of potassium oleate and lithium gamma-linolenate at 299 Ii in a pH 7.0 phosphate buffer. Cyclic voltammetric measurements showed a significant decrease in the surface charge density due to oxide formation on the electrode surface in an aqueous solution in the presence of the lipids, A lipid barrier was formed that was impermeable to the transport of water and azide ions to the electrode surface. The antibiotic ionophores, nystatin, a polyene which transports primarily anions across membranes, and alamethicin, an antibiotic peptide which transports anions and excludes cations, were introduced into the SALL in separate experiments. The surface charge density increased in the presence of these ionophores as a result of diffusion of water to the electrode surface through the apparent ionophore pores. Azide ions diffused through the ionophore-incorporated SALL and were detected and measured electrochemically by a decrease in surface charge density from oxide due to the blocking of the electrode surface by the adsorbed azide ions. The electrode surface coverage was determined to be 16.4 +/- 0.4 water molecules or 15.6 +/- 1.8 azide ions per nystatin pore and 57.2 +/- 1.1 water molecules or 56.6 +/- 3.8 azide ions per alamethicin pore, These results agree with calculations for monolayer coverage based on geometrical dimensions of the molecules.