Production of 2-ketogluconic acid in batch fermentation was investigated. Acetobacter pasteurianus ATCC 6438, which produces selectively 2-ketogluconic acid only, was used. The optimal pH for glucose dehydrogenation to gluconate by resting cells was 5.0 and for gluconate dehydrogenation to 2-ketogluconate 4.25. When glucose medium was used, the 89% yield was achieved after 90 h. For the optimal productivity, medium containing glucose and gluconate with the molar glucose:gluconate ratio 7.4 was proposed, and the yield of 92% after 56 h was achieved. This composition of medium led to the elevation of dissolved oxygen concentration during fermentation. It consequently resulted in elevated gluconate dehydrogenase activity being discussed as the rate-limiting activity of the batch production.