The R2 subunit of Escherichia coli ribonucleotide reductase (RNR) contains a cofactor which consists of a stable tyrosyl radical (*Y122) adjacent to a mu-oxo-bridged diiron(III) cluster. This cofactor assembles spontaneously when apo R2 is treated with Fe2+ and O-2. By using rapid kinetic techniques, two kinetically competent intermediates in this assembly process were recently identified (Bollinger, J. M., Jr. et al. Science 1991, 253, 292-298). One of the intermediates is a paramagnetic iron species. By using the rapid freeze-quench technique and a mutant R2 subunit in which Y122 is replaced with F, 1 equiv of this intermediate per R2 subunit can be trapped. Previous electron paramagnetic resonance (EPR) and Mossbauer studies suggested that this species isa spin coupled system involving two high-spin ferric ions and a free radical (a "diferric radical species") (Bollinger, J. M., Jr. et al. J. Am. Chem. Sec. 1991, 113, 6289-6291). In the present study, Mossbauer spectra have been recorded over a wide range of applied fields (60 mT-8 T), and detailed analysis of the Mossbauer data consolidates the initial finding. The observed isomer shifts (0.55 mm/s for iron site 1 and 0.36 mm/s for site 2) and quadrupole splittings (similar to 1 mm/s) are typical for high-spin ferric species. The magnetic hyperfine coupling tensors, A, for the two iron sites are relatively isotropic (within 20%), a feature which is unique to high-spin Fe(III). By using a simple three-spin coupling model (S-1 = 5/2 and S-2 = 5/2 for the two iron sites, and S-3 = 1/2 for the radical), both the signs and magnitudes of the observed A values for the iron sites (A(1)/g(m) beta(n) = -52.5 T and A(2)/g(m) beta(n) = +24 T) can be explained with an intrinsic A value that is consistent with high-spin Fe(III). The asymmetry in the A values for the two iron sites is a result of the spin orientations : the Spin of the radical is parallel to that of the negative-A-value Fe site and antiparallel to that of the positive-A-value Fe site. Since it has been suggested on the basis of functional analogy with heme-iron-dependent peroxidases that generation of *Y122 in RNR-R2 may involve a high-valent iron-oxo intermediate, the possibility that this paramagnetic iron species contains Fe(IV) has also been carefully examined. Both the EPR and the Mossb;auer data are incompatible with such a possibility.