A sensitive method for measuring current-voltage (IV) characteristics of the type I( U) ≈ χ U(1 + β U 2) was employed to study the influence oflipid composition and cholesterol on ionic transport through gramicidin D channels. The nonlinearity coefficient f) of the IV characteristics depended on the electrolyte concentration c, the lipid composition and the cholesterol content. The value of fi was proportional to log( c) for different phospholipids. Considerable differences in β were found for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and choline plasmalogen bilayer lipid membranes (BLMs) at relatively low ionic strengths ( c ≈ 0.01–0.03 M KCl). In this range β < 0 and its absolute value was less for the POPC BLM than for choline plasmalogen. Considerable differences were also found between zwitterionic phospholipids and negatively charged soybean phosphatidylcholine (SBPC) in the concentration range tested (0.01–3 M). Also, the slope of the graph of β versus log( c) was less for SBPC than for the other phospholipids. Almost no differences in the dependence of β on log( c) were found between chargeless lipids (glycerolmonooleate(GMO)) and zwitterionic phospholipids. The addition of 33 mol% cholesterol to egg phosphatidylcholine (eggPC) leads to a decrease in the slope of β versus log( c); at a higher cholesterol concentration (66 mol%) this slope changes sign and 18 > 0 at all electrolyte concentrations. The value of β correlates well with the order parameter S determined by spin probe I 12,3 as a function of cholesterol concentration. Presentation of experimental data in terms of the profile of standard electrochemical potential and ion-channel macromolecule shape in the framework of an electrodiffusion model of ion transport allows the influence of lipid composition and cholesterol on the gramicidin channel to be explained.