The molecular mechanism of oxidative deposition of iron in ferritin is incompletely understood. In this study, EPR-active species produced during ferritin reconstitution (10-50 Fe/protein) from the apoprotein, Fe2+, and O2 have been investigated using rapid-mixing freeze-quench techniques and EPR spectroscopy. Species studied include a monomeric Fe(3+)-protein complex (g' = 4.3), a mixed-valent Fe(2+)-Fe3+ complex (g' = 1.87), and a newly observed radical with axial symmetry (g parallel = 2.042, g perpendicular = 2.0033), all apparent intermediates formed during the first second of iron oxidation. The monomeric Fe(3+)-protein complex is the principal EPR-observable product of iron(II) oxidation and is produced quantitatively in the first phase of the reaction with the mixed-valent species and the radical formed at slower rates. The initial rate of formation of the monomeric complex (and the radical) is first-order in Fe2+ concentration, consistent with a mechanism in which iron oxidation occurs in a one-electron step(s) with H2O2 being the final product of O2 reduction. A 1:1 relationship between the disappearance of the monomeric Fe(3+)-protein complex and the formation of the mixed-valent Fe(2+)-Fe3+ species was observed in the early phase of the reaction, indicating that the latter is derived from the former and not from the one-electron oxidation of a preformed Fe(2+)-Fe2+ dimer. The g-factors and rapid EPR relaxation properties of the transient radical suggest that it is associated with an Fe2+ (or Fe3+) center but its identity and possible functional role in iron oxidation are unknown.