Selective H-2-, C-13-, and O-17-isotope labeling of the tyrosine amino acid has been used to map the unpaired pi-electron spin-density distribution of the UV-generated neutral L-tyrosine phenoxy radical in alkaline frozen solution. The use of C-13 and O-17 labels allowed accurate determination of the full spin-density distribution and provided more insight in the geometrical structure of the neutral tyrosine radical in vitro. Simulations of the X-band (9.2 GHz) and Q-band (34.8 GHz) EPR powder spectra yielded the principal components of the H-1-, C-13-, and O-17-hyperfine tensors. For the two beta-methylene hydrogens, a static conformational distribution of the dihedral angles (90 degrees < theta(1) < 60 degrees and 60 degrees < theta(2) < 30 degrees) was taken into account. The major proton hyperfine interactions and the principal g values for the neutral tyrosine radical, obtained from selectively deuterated samples, are consistent with literature values. The spin density at the specifically labeled postitions (C1', C2', C3', C4', C5', O4') was evaluated from the anisotropy of the C-13-and O-17-hyperfine tensors. A quantitative analysis of the positions C3' and C5' provided evidence for a planar distortion of the aromatic ring at these positions. O-17 enrichment of the phenol oxygen O4' of the tyrosine radical unambiguously showed that the spin density at this oxygen is 0.26 +/- 0.01. From the relatively large delocalization of the spin density over the carbonyl group of the tyrosine aromatic ring system, it is concluded that the C4'-O4' bond has a double-bond character. The experimentally determined spin-density distribution is compared with several computational calculated spin-density distributions found in the literature. The isotropic C-13-hyperfine interactions are discussed in the framework of the Karplus-Fraenkel theory. This theory proved to be accurate for the determination of sign and magnitude of the isotropic C-13-and O-17-hyperfine interactions.