The spin density distribution of the Y-122 tyrosyl radical in the R2 subunit of ribonucleotide reductase from Escherichia coli has been determined. Incorporation of isotopically labeled tyrosine into the protein has allowed us to measure the O-17 hyperfine coupling by using EPR, giving a direct measure of the tyrosine phenol oxygen spin density, 0.29 +/- 0.02. The hyperfine tensors of six protons of the radical have been determined by using ENDOR. Magnetic field selection allows a determination of the orientation of the hyperfine tensors relative to the g tenser. Electron-nuclear-nuclear triple resonance has been applied to establish the relative signs of three hyperfine couplings. These measurements give a more precise and more accurate picture of the spin density distribution in a protein tyrosyl radical than has been available previously. The O-17 hyperfine splitting in tyrosyl radicals in aqueous glasses has also been measured. The differences in hyperfine couplings indicate that addition of a hydrogen bond to the phenolic oxygen perturbs the spin density in the ring slightly and causes the spin density at the oxygen atom to decrease by about 10%. Comparison of our results for the ribonucleotide reductase Y-122 tyrosyl radical with those for other naturally occurring tyrosyl radicals and with tyrosines in aqueous glasses shows that there is only slight variation in spin density distribution over the phenol ring in this class of radicals, despite substantial variation in local environment.