Protocatechuate 3,4-dioxygenase (EC 1.13.1.3) was purified to homogeneity from extracts of Azotobacter vinelandii. The molecular weight of the oligomeric protein was estimated to be 510 000 by gel filtration and 480 000 by ultracentrifugation. The oligomer appears to be formed by association of equal amounts of nonidentical subunits which were estimated by sodium dodecyl sulfate gel electrophoresis to have respective molecular weights of 23 300 and 25 250. Ten gram-atoms of iron was associated with each mol of oligomer. Therefore, the enzyme appears to be a decamer with the structure 10(alpha beta Fe). T-HE AMINO ACID COMPOSITION OF Azotobacter protocatechuate oxygenase closely resembles the amino acid compositions of protocatechuate 3,4-dioxygenases from Pseudomonas aeruginosa and Thiobacillus sp. These proteins from P. aeruginosa and P. putida are known to be formed by association of nonidentical subunits of a physical size similar to the subunits of the Azotobacter enzyme. Furthermore, antisera prepared against the Azotobacter oxygenase cross-reacted strongly with the isofunctional enzymes from the two fluorescent Pseudomonas species. A weak immunological cross-reaction was observed when the antisera were tested against protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus. The results favor the conclusion that the bacterial protocatechuate 3,4-dioxygenases were derived from a common ancestral protein.