O-Pyrocatechuic acid (2,3-dihydroxybenzoic acid) carboxylyase (E.C. 4.1.1.), enzyme involved in the biosynthesis of catechol by Aspergillus niger, has been extracted from the acetone powders of mycelial mats grown in the presence of l-tryptophan and purified 815-fold by precipitation of nucleic acids, fractionation with ammonium sulfate and acetone, negative adsorption on hydroxylapatite, and nitration through Sephadex G-100. The purified enzyme is a simple protein without any prosthetic group. It acts specifically on o-pyrocatechuic acid and produces stoichiometric quantities of catechol and carbon dioxide. The enzyme is most active around pH 5.2 and 45 °. The K m value is 0.3 mm. In contrast to other known aromatic carboxy-lyases, this enzyme does not show a requirement for any cofactor or metal ion. Sulfhydryl groups are involved in the reaction. Structural analogs like salicylic acid competitively inhibit the carboxy-lyase activity.