The reactions of free and DNA-bound 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) probes with radicals generated during radiolysis of dilute aqueous solutions of DNA were examined. For the free PROXYL probe in deaerated solution with each of the four nucleotides (dAMP, dCMP, dGMP, and TMP) it was found that the pyrimidine radicals were more reactive toward the probe than were the purine radicals. Reactions of the electron adduct of TMP and the hydroxyl radical adducts of dAMP, dGMP, and TMP with the probe resulted in little or no reduction of the probe. For TMP these results are consistent with the fact that both the protonated electron and hydroxyl radical adducts of TMP will covalently bind to the nitroxide function of the probe. Reduction of the PROXYL probe was observed in reactions with the hydroxyl radical adduct of dCMP and with the electron adducts of dAMP, dCMP, and dGMP. Results of the radiolysis of the free PROXYL probe in deaerated dilute solution of DNA suggest that the PROXYL probe protects the DNA from water radical attack as the ratio of DNA bases to PROXYL probe increases above 50:1. Reactions of DNA-bound probes are dependent on the depth of the nitroxide function in relation to the major groove of the DNA helix. Two probes with tether lengths which are less than the depth of the major groove show an expected increase in reactions with DNA base radicals as compared to a probe with a tether that extends beyond the groove. The longer probe is involved largely in reactions with sugar and water radicals along the periphery of the DNA helix. In the presence of oxygen, there is a dramatic decrease in the loss of both the free and DNA-bound probes due to the lack of reaction of these probes with peroxyl radicals formed by the addition of molecular oxygen to DNA radicals.