A mixed culture of two Gram-negative bacteria isolated from soil converted 50 g D-glucose l(-1) to 2,5-diketo-D-gluconate (2,5 DKG) in 92% yield within 150 h. The first strain, producing 2-keto-D-gluconate (2 KDG) from d-glucose via d-gluconate (DG), was classified as Flavimonas oryzihabitans. The second strain, that converts 2 KDG to 2,5 DKG, was identified as Pseudomonas cepacia. This approach presents a new possibility to produce ascorbic acid by microbial transformation, including the use of other, more convenient substrates.