Three isozymes of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas arvilla C-1 were separated using DEAE-Toyopearl chromatography. The specific activities of each isozyme were similar to one another. The molecular weights of isozymes 1, 2, and 3 were estimated to be approximately 67,000, 64,000, and 59,000, respectively, from gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isozymes 1 and 3 gave a single protein band, corresponding to Mr = 32,000 and 30,000, respectively, and isozyme 2 gave two bands corresponding to Mr = 32,000 and 30,000. These results indicated that isozymes 1 and 3 were homodimers, while isozyme 2 was a heterodimer. The NH2-terminal sequences up to 20 residues of these three isozymes confirmed that isozymes 1, 2, and 3 consisted of beta beta, alpha beta, and alpha alpha, respectively, based on our previous data (Nakai, C., Kagamiyama, H., Saeki, Y., and Nozaki, M. (1979) Arch. Biochem. Biophys. 195, 12-22). Properties of these isozymes such as absorption spectrum, iron content, substrate specificity, and kinetic constants were similar to one another. Subunit exchange between the different isozymes and dissociation of the isozymes into subunits was not observed under nondenaturing conditions. Available evidence indicates that these isozymes exist naturally in the bacterium and were not due to artifacts caused by purification.