This work reports ESR studies that identify the favored site of deprotonation of the guanine cation radical (G(center dot+)) in an aqueous medium at 77 K. Using ESR and UV-visible spectroscopy, one-electron oxidized guanine is investigated in frozen aqueous D2O solutions of 2'-deoxyguanosine (dGuo) at low temperatures at various pHs at which the guanine cation radical, G(center dot+) (pH 3-5), singly deprotonated species, G(-H)(center dot) (pH 7-9), and doubly deprotonated species, G(-2H)(center dot-) (pH > 11), are found. C-8-deuteration of dGuo to give 8-D-dGuo removes the major proton hyperfine coupling at C-8. This isolates the anisotropic nitrogen couplings for each of the three species and aids our analyses. These anisotropic nitrogen couplings were assigned to specific nitrogen sites by use of N-15-substituted derivatives at N1, N2, and N3 atoms in dGuo. Both ESR and UV-visible spectra are reported for each of the species: G(center dot+), G(-H)(center dot), and G(-2H)(center dot-). The experimental anisotropic ESR hyperfine couplings are compared to those obtained from DFT calculations for the various tautomers of G(-H)(center dot). Using the B3LYP/6-31G(d) method, the geometries and energies of G(center dot+) and its singly deprotonated state in its two tautomeric forms, G(N1-H)(center dot) and G(N2-H)(center dot), were investigated. In a nonhydrated state, G(N2-H)(center dot) is found to be more stable than G(N1-H)(center dot), but on hydration with seven water molecules G(N1-H)(center dot) is found to be more stable than G(N2-H)(center dot). The theoretically calculated hyperfine coupling constants (HFCCs) of G(center dot+), G(N1-H)(center dot), and G(-2H)(center dot-) match the experimentally observed HFCCs best on hydration with seven or more waters. For G(-2H)(center dot-), the hyperfine coupling constant (HFCC) at the exocyclic nitrogen atom (N2) is especially sensitive to the number of hydrating water molecules; good agreement with experiment is not obtained until nine or 10 waters of hydration are included.