The maganese-containing superoxide dismutase, from Escherichia coli, lost metal and activity when dialyzed against 20 mM-8-hydroxyquinoline and 2.5 M guanidinium chloride in 5 mM Tris-chloride buffer at pH 7.8. Subsequent dialysis against 0.01 m M McCl2, in this buffer, caused a restoration of manganese and activity. Reconstituted enzyme appeared identical with native enzyme, and removal and restoration of manganese could be repeated. Co (II), Zn(II), Ni(II), Mg(II), Cr (II), Cu(II), Fe(II), In(II), and Mo(VI), were tested for their abiltiy to replace manganese. None of these restored activity to apoenzyme. When present at a 100-fold molar excess over manganese only Co(II), Ni(II), and Zn(II) were effective as competitors of manganese. Co(II) was demonstrated to be tightly bound to the apoenzyme in place of the magnasese, with a stoichiometry of 1 Co(II) per molecule. In the cases of Co(II), Zn(II), or Ni(II), reconstituted enzyme; the metals could be removed and subsequently replaced by manganese, with restoration of catalytic activity. None of these metal cations exhibited superoxide dismutase activity. We conclude that manganese is essential for the catalytic activity of superoxide dismutase, and that the site which normally binds manganese can accommodate Co(II), Ni(II), and Zn(II).