A novel 55-kDa hydroxylase was isolated from cultured cells of Catharanthus roseus by a three-step procedure: anion exchange chromatography, affinity chromatography and hydroxylapatite adsorption chromatography. The enzyme specifically catalyzed the hydroxylation of 2-hydroxy-benzoic acid to give 2,5-dihydroxybenzoic acid. The enzyme activity was optimal at pH 7.8 and was completely inhibited by divalent cations, such as Cu2+ and Hg2+. The enzyme showed sequence similarity to certain plant flavonoid 3'-hydroxylases.