Two different tryptophan radicals (W-a(.) and W-b(.)) with lifetimes of several minutes at room temperature are formed during the reconstitution of the diiron center in the Escherichia coli ribonucleotide reductase mutant protein R2 Y122F. Detailed hyperfine parameters are for the first time determined for protein-linked oxidized neutral tryptophan radicals. Wa(.) is freeze-trapped and investigated by EPR and ENDOR in protonated and selectively deuterated proteins at 20 K. Two hyperfine couplings from the beta-methylene protons, hyperfine tensors of two alpha-protons, and the complete nitrogen hyperfine tensor are determined. Based on the absence of a large hyperfine coupling from the N-H proton, which would be expected for a cation radical, and on comparison of the experimental data with theoretical spin densities from density functional calculations, W-a(.) is assigned to an oxidized neutral tryptophan radical. A small anisotropic hyperfine coupling detected in selectively deuterated Wa is tentatively assigned to a proton which is hydrogen bonded to the nitrogen of W-a(.). A similar spin density distribution as for W-a(.) is obtained also for the second tryptophan radical, W-b(.) observed by EPR at room temperature, which is also assigned to an oxidized neutral radical.