Ion channels, such as gramicidin A, selectively facilitate the transport of ions across biological and synthetic membranes. The conductance properties of ion channels are frequently characterized in synthetic bilayer lipid membranes (BLMs). The instability of BLMs has seriously limited the range of applications for these structures, and tethered bilayer lipid membranes (tBLMs) have addressed the problem through tethering many of the membrane components to a solid surface. In the present study, thin gold substrates have been used to tether thiol- and disulfide-terminated membrane components to form a tBLM electrode to provide a reservoir for ions. This study reports on the ion selectivity and apparent permeability of gramicidin channels in such tethered bilayer membranes. The investigations using electrical impedance spectroscopy indicated that the magnitude of ionic conductance varies substantially in reservoirs with different chemical structures. This study addressed the effect of changing ionic concentration, the effect of changing the species in the bulk solution above the membrane, and the influence of the chemical structure of the reservoir tethers. The effect of two-dimensional packing on membrane conductance was also investigated. The present observations suggested that (a) the reservoir region resistivity has a major influence on the overall conductivity of the membrane and in some instances can dominate conduction, (b) the conduction behavior is nonlinear and exhibits saturation with increasing electrolyte concentration, and (c) that ion pairing in the reduced dielectric (epsilon similar to50) reservoir region is the likely basis for the latter effect. The inferred limiting ionic mobilities of alkali chloride species in the membrane reservoir regions were 3-4 orders of magnitude less than in aqueous solution, indicating that the reservoirs resembled hydrated polymer gels.