Intracellular microelectrode techniques were employed to study the mechanism by which amphotericin B induces a transient mucosa-negative transepithelial potential (deltaVms) in the gallbladder of Necturus. When the tissue was incubated in standard Na-Ringer's solution, the antibiotic reduced the apical membrane potential by about 40 mV, and the basolateral membrane potential by about 35 mV, whereas the transepithelial potential increased by about 5 mV. The electrical resistance of the apical membrane fell by 83%, and that of the basolateral membrane by 40%; the paracellular resistance remained unchanged. Circuit analysis indicated that the equivalent electromotive forces of the apical and basolateral membranes fell by 35 and 11 mV, respectively. Changes in potentials and resistances produced by ionic substitutions in the mucosal bathing medium showed that amphotericin B produces a nonselective increase in apical membrane small monovalent cation conductance (K, Na, Li). In the presence of Na-Ringer's on the mucosal side, this resulted in a reduction of the K permselectivity of the membrane, and thus in a fall of its equivalent emf. During short term exposure to amphotericin B, PNa/PCl across the paracellular pathway did not change significantly, whereas PK/PNa doubled. These results indicate that deltaVms is due to an increase of gNa across the luminal membranes of the epithelial cells (Cremaschi et al., 1977. J. Membrane Biol. 34:55); the data do not support the alternative hypothesis (Rose & Nahrwold, 1976. J. Membrane Biol 29:1) that deltaVms results from a reduction in shunt PNa/PCl acting in combination with a rheogenic basolateral Na pump.