Pyridine-2,3-dicarboxylic acids are prepared by the action of 2,3-dihydroxybenzoate-3,4-dioxygenase on 2,3-dihydroxybenzoic acids in a liquid medium which lacks active decarboxylase and which has a pH from 4 to 9, an ionic strength below about 1 molar, and a low concentration of metal cations and complex anions. In close temporal proximity, the 2-hydroxy-3-carboxymuconic acid semialdehyde which forms is allowed to react with a source of ammonia or a primary amine, avoiding substantial decarboxylation.