The obligately fermentative aerotolerant bacterium Zymomonas mobilis was shown to possess oxidative phosphorylation activity. Increased intracellular ATP levels were observed in aerated starved cell suspension in the presence of ethanol or acetaldehyde. Ethanolconsuming Z. mobilis generated a transmembrane pH gradient. ATP synthesis in starved Z. mobilis cells could be induced by external medium acidification of 3.5–4.0 pH units. Membrane vesicles of Z. mobilis coupled ATP synthesis to NADH oxidation. ATP synthesis was sensitive to the protonophoric uncoupler CCCP both in starved cells and in membrane vesicles. The H+-ATPase inhibitor DCCD was shown to inhibit the NADH-coupled ATP synthesis in membrane vesicles. The physiological role of oxidative phosphorylation in this obligately fermentative bacterium is discussed.