The effect of known precursors and cofactors on vitamin B12 production has been studied in a basal, chemically defined medium composed of sucrose, glutamic acid, and mineral salts. Only a carbon source, betaine, and Co2+ were found to be essential for vitamin B12 production. Known precursors such as methionine, δ-aminolevulinic acid, succinic acid, and 1-amino-2-propanol had no effect. Glycine was inhibitory to growth. Oxalic acid, lactic acid, 5,6-dimethylbenzimidazole, and Mo2+ stimulated vitamin B12 biosynthesis. Of special interest is oxalic acid, which at a level of 0.04% w/v increased vitamin production about 20%, and with washed cells could replace glutamic acid, which had been demonstrated to be essential for both growth and vitamin B12 production. When use of oxalate was followed, it was found that this acid disappeared during the period of rapid growth and vitamin production and then reappeared during the final stages of the fermentation cycle.