The mechanism of inhibition of active Cl- secretion by 1 mM furosemide and 0.1 mM bumetanide was characterized in the isolated frog corneal epithelium. Transepithelial and transmembrane cell electrical parameters as well as transmembrane Cl- electrochemical potential difference were measured with conventional glass microelectrodes and Cl- selective microelectrodes. Furosemide caused the potential difference across the apical membrane to hyperpolarize by 20 mV while the transepithelial potential difference declined by 13 mV. The apical-to-basolateral membrane resistance ratio increased 3-4 times after furosemide or bumetanide addition. Preincubation with furosemide prevented a 30-mV depolarization of the apical membrane potential difference normally observed when Cl- was removed from the tear side bathing solution. In control conditions, intracellular Cl- activity was above equilibrium. Bumetanide further increased the Cl- electrochemical gradient between the cell compartment and the bathing solutions even though intracellular Cl- activity fell from 18 to 12 mM. In contrast, perfusion with Cl- -free Ringer in the stromal side bathing solution decreased the Cl- electrochemical gradient across the apical membrane to zero, indicating an equilibrium distribution. Adenosine, which selectively increases Cl- permeability of the apical membrane, also decreased the Cl- electrochemical gradient across the apical membrane. These results suggest that the diuretics inhibit active Cl- transport primarily by decreasing the Cl- permeability of the apical membrane.