Abstract The presence of dinitrophenol (DNP) during the chemostatic growth of Acetobacter methanolicus on glucose led to i) no significant increase in glucose dehydrogenase, ii) a decrease in the capacity to oxidize glucose by about 30%, not depending on the DNP concentration, and iii) a gradual decrease in the capacity to form gluconic acid, depending on the concentration of the uncoupler. Similar effects of DNP on Acetobacter methanolicus were observed during the gluconic acid formation process. The loss in the capacity to form gluconic acid is indicated by a drop in the ATP concentration of the cells which cannot be counteracted by the ATP syntheses originating from the oxidation of gluconic acid and of glucose. ATP could be necessary for pumping protons out of the cells that were taken up together with gluconic acid and by the effect of DNP.