Escherichia coli B, grown under aerobic conditions, contains at least three distinct superoxide dismutases, which can be visualized on polyacrylamide gel electropherograms of crude soluble extracts of the sonically disrupted cells. Of these, the slowest migrating and the fastest migrating, respectively, have previously been isolated and characterized as manganese-containing and iron-containing enzymes. The enzyme form with medium electrophoretic mobility has now been purified to homogeneity. Its molecular weight is approximately 37,000 and it contains 0.8 atoms of iron/molecule and only negligible amounts of manganese. Like other iron-containing superoxide dismutases and unlike the corresponding manganienzymes, it is inactivated by EDTA plus H2O2. Its specific activity is comparable to that of the other superoxide dismutases of E. coli. Two types of subunits could be distinguished upon electrophoresis in the presence of sodium dodecyl sulfate. One of these migrated identically with the subunit obtained from the manganisuperoxide dismutase, while the other similarly appeared identical with the subunit from the ferrisuperoxide dismutase. This newly isolated enzyme thus appears to be a hybrid of the other two forms. In support of this conclusion, we observed that ultrafiltration or storage of the new superoxide dismutase gave rise to the mangani- and ferrienzymes on disc gel electrophoresis or isoelectric focussing.