Apical plasma membrane vesicles isolated from bovine tracheal epithelium were found to possess both an electrical potential gradient (delta psi) and a transmembrane pH gradient (delta pH). The delta psi was calculated from the distribution of 86Rb+ in the presence of the ionophore valinomycin, and delta pH was determined from the distribution of [14C]methylamine under conditions of apparent equilibrium. Maximal values for delta psi of -54.3 +/- 2.5 mV and for delta pH of 0.75 +/- 0.07 pH units were obtained under low ionic strength conditions. Increasing the ionic strength by the addition of 50 mM of either permeant or impermeant electrolytes reduced delta psi to near zero values. The delta pH varied in parallel with the delta psi. The results suggest that both 86Rb+ in the presence of valinomycin and H+ are in a Donnan equilibrium with impermeant negative charge in the vesicle interior.