A practical method for the production of calcium 2-keto-l-gulonate (an intermediate in the Reichstein synthesis of l-ascorbic acid) from d-glucose has been established by using a two-stage fermentation system. d-Glucose was first converted to calcium 2,5-diketo-d-gluconate by a mutant strain of Erwinia sp. in a medium containing d-glucose, corn steep liquor, (NH(4))(2)HPO(4), and CaCO(3). After a 26-h cultivation, 328.6 mg of calcium 2,5-diketo-d-gluconate per ml was obtained, with a 94.5% yield from d-glucose. This broth was used directly for the next conversion without removal of cells by treatment with sodium dodecyl sulfate. The stereospecific reduction of calcium 2,5-diketo-d-gluconate to calcium 2-keto-l-gulonate was performed with a mutant strain of Corynebacterium sp. When the cell growth reached a maximum (about 16 h) in a medium containing d-glucose, corn steep liquor, NaNO(3), KH(2)PO(4), and trace elements, NaNO(3) was added to the culture, and then the calcium 2,5-diketo-d-gluconate broth was fed over a period of about 50 h. Since the mutant strain requires a hydrogen donor for reduction, the calcium 2,5-diketo-d-gluconate broth was mixed with d-glucose before being fed. The results of four two-stage fermentations in 10-m conventional fermentors showed that an average of 106.3 mg of calcium 2-keto-l-gulonate per ml was obtained, with a 84.6% yield from d-glucose, the starting material of calcium 2,5-diketo-d-gluconate production. Calcium 2-keto-l-gulonate was stable in the broth. Neither 2-keto-d-gluconic acid nor 5-keto-d-gluconic acid was detected in the final broth.