3-Carbamoyl-alpha-picolinic acid, a versatile building block for the synthesis of agrochemicals and pharmaceuticals, was prepared by imidase-catalyzed regiospecific hydrolysis of 2,3-pyridinedicarboximide with intact Arthrobacter ureafaciens O-86 cells. Reactions were carried out in a water-organic solvent, two-phase system containing cyclohexanone at low pH to avoid spontaneous random hydrolysis. Under the optimized conditions, with the periodic addition of 2,3-pyridinedicarboximide (in total, 40 mM), the 3-carbamoyl-alpha-picolinic acid yield reached 36.6 mM in the water phase, with a molar conversion yield of 91.5% and a regioisomeric purity of 94.5%, in 2 h at pH 5.5.