2-Keto- d-gluconate and 2-keto- d-galactonate were prepared from d-glucose (with a yield of 40%) and d-galactose (with a yield of 25%), respectively, with cell culture of Pseudomonas fluorescens. However, 2-keto- d-mannoate was not prepared in this method. The time courses of the reactions showed that 2-keto- d-gluconic acid and 2-keto- d-galactonic acid were produced from d-glucose and d-galactose through d-gluconate and d-galactonate, respectively. When using d-galactono 1,4-lactone as a starting material, 2-keto- d-galactonate was produced with partially purified NADP-dependent 2-ketogalactonate reductase from P. fluorescens. Some fundamental properties of the 2-ketogalactonate reductase were compared with those of 2-ketogluconate reductase from Acetobacter and Gluconobacter.