Inverted membrane vesicles prepared from Escherichia coli ML 308-225 generate a transmembrane electrochemical proton gradient (delta mu H+; interior positive and acid) during oxidation of D-lactate, succinate, reduced phenazine methosulfate, or NADH or hydrolysis of ATP. Using the distribution of the lipophilic anion thiocyanate to measure the membrane potential (delta psi) and the permeant weak base methylamine to measure the pH gradient (delta pH), maximal values for delta psi H+ of approximately +160 mV are obtained. Many of the properties of delta psi H+ in inverted vesicles are similar to those described previously in right-side-out vesicles [Ramos, S., & Kaback, H.R. (1977) Biochemistry 16, 848]: (1) the magnitude of the delta psi (interior positive) generated in the presence of D-lactate or reduced phenazine methosulfate is similar to that observed in right-side-out vesicles but of opposite polarity and independent of pH from 5.5 to 8.0; (2) plots of delta pH vs. internal pH in the right-side-out vesicles are similar with D-lactate as the electron donor; (3) as observed with right-side-out vesicles, dissipation of delta psi or delta pH leads to a concomitant increase in the other parameter without a change in the rate of respiration; (4) inverted vesicles catalyze Na+ accumulation, and it is apparent that the process can be driven by either delta psi (interior positive) or delta pH (interior acid).