Membrane vesicles prepared from Zymomonas mobilis oxidized NADH exclusively, whereas deamino-NADH was little oxidized. In addition, the respiratory chain-linked NADH oxidase system exhibited only a single apparent Km value of approximately 66 microM for NADH. The NADH oxidase was highly sensitive to the respiratory chain inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide. However, the NADH:quinone oxidoreductase was not sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide and was highly resistant to another respiratory chain inhibitor, rotenone. Electron transfer from NADH to oxygen generated a proton electrochemical gradient (inside positive) in inside-out membrane vesicles. In contrast, electron transfer from NADH to ubiquinone-1 generated no electrochemical gradient. These findings indicate that Z. mobilis possesses only NADH:quinone oxidoreductase lacking the energy coupling site.