Optically black, thin lipid membranes prepared from sheep erythrocyte lipids have a high dc resistance (R(m) congruent with 10(8) ohm-cm(2)) when the bathing solutions contain NaCl or KCl. The ionic transference numbers (T(i)) indicate that these membranes are cation-selective (T(Na) congruent with 0.85; T(Cl) congruent with 0.15). These electrical properties are independent of the cholesterol content of the lipid solutions from which the membranes are formed. Nystatin, and probably amphotericin B, are cyclic polyene antibiotics containing approximately 36 ring atoms and a free amino and carboxyl group. When the lipid solutions used to form membranes contained equimolar amounts of cholesterol and phospholipid, these antibiotics reduced R(m) to approximately 10(2) ohm-cm(2); concomitantly, T(Cl) became congruent with0.92. The slope of the line relating log R(m) and log antibiotic concentration was congruent with4.5. Neither nystatin (2 x 10(-5)M) nor amphotericin B (2 x 10(-7)M) had any effect on membrane stability. The antibiotics had no effect on R(m) or membrane permselectivity when the lipids used to form membranes were cholesterol-depleted. Filipin (10(-5)M), an uncharged polyene with 28 ring atoms, produced striking membrane instability, but did not affect R(m) or membrane ionic selectivity. These data suggest that amphotericin B or nystatin may interact with membrane-bound sterols to produce multimolecular complexes which greatly enhance the permeability of such membranes for anions (Cl(-), acetate), and, to a lesser degree, cations (Na(+), K(+), Li(+)).