The deprotonation of guanine cation radical (G(+center dot)) in oligonucleotides (ODNs) was measured spectroscopically by nanosecond pulse radiolysis. The G(+center dot) in ODN, produced by oxidation with SO4-center dot, deprotonates to form the neutral G radical (G(-H)(center dot)). In experiments using 5-substituted cytosine-modified ODN, substitution of the cytosine C5 hydrogen by a methyl group increased the rate constant of deprotonation, whereas replacement by bromine decreased the rate constant. Kinetic solvent isotope effects on the kinetics of deoxyguanosine (dG) and ODN duplexes were examined in H2O and D2O. The rate constant of formation of G(-H)(center dot) in dG was 1.7-fold larger in H2O than D2O, whereas the rate constant in the ODN duplex was 3.8-fold larger in H2O than D2O. These results suggest that the formation of G(-H)(center dot) from G(+center dot) in the ODN corresponds to the deprotonation of the oxidized hydrogen-bridged (G'*-C) base pair by a water molecule. The characteristic absorption maxima of G(+center dot) around 400 nm were shifted to a longer wavelength in the order of G < GG < GGG-containing ODNs. In contrast, the spectra of G(-H)(center dot) were not affected by the sequence and were essentially similar to that of free dG. These results suggest that the positive charge in G(+center dot) in ODN is delocalized over the extended pi orbitals of DNA base. The rate constant of the deprotonation was altered by the sequence of ODNs, where bases adjacent to guanine are important factors for deprotonation.