Intracellular microelectrode techniques were employed to study the effects of diphenylamine-2-carboxylate (DPC) on ion transport in Necturus gallbladder epithelium. Under control conditions, addition of DPC to the mucosal bathing solution caused a concentration-dependent, reversible hyperpolarization of both cell membranes with no measurable resistance changes. In addition, DPC caused the following effects, all consistent with inhibition of apical membrane Cl(-)-HCO3- exchange: fall in intracellular Cl- activity (aCli), increase in intracellular pH (pHi), reduction of the changes in aCli and pHi produced by lowering mucosal solution [Cl-], and reduction of the change in pHi produced by lowering mucosal solution [HCO3-]. Similar studies in theophylline-treated preparations indicate that DPC also inhibits anion exchange under these conditions, but has no effect on the apical membrane electrodiffusive Cl- permeability induced by cyclic AMP. Under these conditions, DPC caused cell membrane hyperpolarization but had no effect on the apparent ratio of membrane resistances. In addition, DPC had no effects on the rapid changes in apical membrane voltage elicited by altering mucosal [Cl-], but caused significant reductions of the slower, secondary voltage changes observed in response to changes in mucosal [Cl-], and the changes in aCli and pHi produced by lowering mucosal [Cl-]. Because others have demonstrated that DPC blocks Cl- channels in other epithelia (Distefano, A., M. Wittner, E. Schlatter, H. J. Lang, H. Englert, and R. Greger. Diphenylamine-2-carboxylate, a blocker of the Cl(-)-conductive pathway in Cl(-)-transporting epithelia. Pfluegers++ Arch. 405: S95-S100, 1985), it is possible that the structures of those channels and that induced by cyclic AMP in Necturus gallbladder are different. Because of its relatively high affinity and rapid reversibility, DPC may become useful in studies of anion exchange in other cells.