Electron spin resonance (ESR) spectroscopy has been used to investigate irreversible protonations of the nucleobase anions in gamma-irradiated frozen aqueous solutions of dGMP-dCMP, polyG-polyC, poly[dGdC].poly[dGdC], dAMP-dTMP, poly[dAdT].poly-[dAdT] and DNA itself. Analysis of the ESR spectra at a dose of 22 kGy shows that fractional conversion of total radicals to carbon-protonated species on annealing is in the order: dAMP-dTMP (43%) > pdAdT = DNA (23%) > dGMP.dCMP (15%) > poly-dGdC.polydGdC (6%) > polyG.polyC (3%). Two hydrogen addition radicals make contributions to the polyG.polyC, poly-[dGdC].poly[dGdC] and dGMP.dCMP spectra in H2O on annealing. They are those formed by protonations at C6 of the cytosine anion radical, C(C6)H., and at C8 of the guanine anion radical, G(C8)H.. Computer analysis reveals that anion protonation reaction in dGMP.dCMP results in mainly C(C6)H., whereas protonation reaction in polyG.polyC and poly[dGdC].poly-[dGdC] yields mainly G(C8)H.. In dAMP.dTMP and poly[dAdT].poly[dAdT] as in DNA itself, the only DNA base found to undergo an irreversible protonation at a carbon site is thymine, resulting in T(C6)H.. The conversion of DNA anion to T(C6)H. is found to be dependent on dose. At low doses (5 kGy), about 30% conversion to T(C6)H. is found, whereas at high doses (94 kGy), only 13% conversion is found. The dose dependence is ascribed in part to ion radical recombinations whose probabilities are increased at high doses. A consideration of the rates of protonations of the purine and pyrimidine anion radicals as well as the differences in electron affinities suggest carbon protonation reactions of DNA base anions in irradiated stacked double-strand DNA at 37 degrees C would be predominantly at thymine and perhaps guanine, whereas in single-strand DNA all bases would contribute.