During growth of Hydrogenomonas eutropha H 16 on 2-ketogluconate, 2-ketogluconate kinase and 2-keto-6-phosphogluconate reductase were formed. These enzymes were absent from cells grown on fructose, gluconate, acetate, succinate or autotrophically. There was no evidence for extracellular oxidation of glucose, fructose or gluconate with the formation of ketogluconic acids. The utilization of 2-ketogluconate is neither subject to catabolite inhibition by hydrogen nor is 2-keto-6-phosphogluconate reductase inhibited by ATP, ADP or phosphoenolpyruvate. The enzyme is characterized by a high affinity for its substrates.