1. Protocatechuate 3,4-dioxygenase (protocatechuate : oxygen 3,4-oxidoreductase, EC 22.214.171.124) was isolated from mycelium of Chaetomium piluliferum induced with p-hydroxybenzoic acid. The enzyme was purified about 80-fold by ammonium sulphate fractionation and DEAE-cellulose and Sephadex G-200 chromatography, and was homogeneous on polyacrylamide-gel electrophoresis. 2. The enzyme showed high substrate specificity; its pH optimum was 7.5-8.0, and molecula weight about 76 000 as determined by filtration on Sephadex G-200. The Michaelis constant for protocatechuic acid was 11.1 microM.