We used intracellular microelectrode techniques to study the mechanisms responsible for Cl secretion by canine tracheal epithelium. Tissues were treated with indomethacin (10(-6) M, added to the mucosal solution) to reduce the baseline rate of Cl secretion and then stimulated by addition of epinephrine (10(-6) M) or prostaglandin E1 (10(-6) M) to the submucosal solution. Three conclusions emerged from our findings: First, secretagogues enhance the rate of transepithelial Cl transport primarily by increasing apical membrane Cl permeability, since: (i) stimulation of secretion produced parallel decreases in transepithelial resistance (Rt) and the membrane resistance ratio Ra/Rb, where Ra and Rb refer to the membrane resistance ratio Ra/Rb, where Ra and Rb refer to the resistances of the apical and basolateral membranes; (ii) there was an inverse relation between the short-circuit current and Ra/Rb; (iii) secretagogues depolarized the electrical potential difference across the apical membrane (psi a) and produced an equivalent hyperpolarization of the transepithelial electrical potential difference (psi) t) so that, in the steady-state, the basolateral membrane potential (psi b) was unchanged; and (iv) substitution of sulfate or gluconate for Cl in the bathing solutions prevented secretagogue-induced changes in Rt, Ra/Rb, psi a, and psi t. Second, Cl entry into the cell across the basolateral membrane appears to be electrically-neutral since omission of Cl from the submucosal solution had no effect on psi b and did not decrease Ra/Rb as would be expected if Cl entered the cell by a conductive process. Third, secretagogues decrease Rb. Approximately 20 sec after the onset of the secretory response Ra/Rb underwent a secondary increase while Rt continued to fall. The decrease in Rb may reflect an increase in basolateral membrane K permeability.