Benzoate-1,2-dioxygenase system from Pseudomonas arvilla C-1 consists of two protein components, benzoate-1,2-dioxygenase reductase and benzoate-1,2-dioxygenase (Yamaguchi, M., and Fujisawa, H. (1980) J. Biol. Chem. 255, 5058-5063). Benzoate-1,2-dioxygenase exhibited two protein bands (alpha and beta) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their molecular weights were estimated to the 50,000 and 20,000, respectively. The intensities of protein staining on polyacrylamide gels suggested that these two subunits were present in equimolar quantities in benzoate-1,2-dioxygenase. Molecular weight of benzoate-1,2-dioxygenase was estimated to be 201,000 by sedimentation equilibrium (Yphantis method). The values of molecular weights of native enzyme and its subunits suggested that the subunit structure of benzoate-1,2-dioxygenase may be alpha 3 beta 3. Cross-linking experiments also suggested the same subunit structure. These two subunits were separated from each other by Ultrogel AcA44 chromatography in the presence of 6 M urea. Amino acid compositions of the two subunits were examined and compared with that of native enzyme. NH2-terminal amino acids of alpha and beta subunits were both serine, and isoelectric points of alpha and beta in the presence of 6 M urea were determined to be pH 5.6 and pH 4.8, respectively. The enzyme contained 8.2 mol of iron and 5.9 mol of labile sulfide/mol of enzyme, suggesting the presence of additional iron atoms besides iron-sulfur clusters. The isolated beta subunit did not contain any significant amounts of iron and labile sulfide, but the alpha subunit contained approximately 2 mol each of iron and labile sulfide and exhibited an absorption spectrum of binuclear iron cluster type.