5-Hydroxypyrazine-2-carboxylic acid, a versatile building block for the synthesis of new antituberculous agents, was prepared by whole-cell biotransformation from 2-cyanopyrazine via pyrazinecarboxylic acid using Agrobacterium sp. DSM 6336. By developing a fermentation process for this two-enzyme-step bioconversion, a product concentration of 286 mM (40 g/l) was obtained. After the isolation method had been optimized the total yield was 80%.