Purine radical cations (dA(center dot+) and dG(center dot+)) are the primary hole carriers of DNA hole migration due to their favorable oxidation potential. Much less is known about the reactivity of higher energy pyrimidine radical cations. The thymidine radical cation (T center dot+) was produced at a defined position in DNA from a photochemical precursor for the first time. T center dot+ initiates hole transfer to dGGG triplets in DNA. Hole localization in a dGGG sequence accounts for similar to 26% of T center dot+ formed under aerobic conditions in 9. Reduction to yield thymidine is also quantified. 5-Formyl-2'-deoxyuridine is formed in low yield in DNA when T center dot+ is independently generated. This is inconsistent with mechanistic proposals concerning product formation from electron transfer in poly(dA-T) sequences, following hole injection by a photoexcited anthraquinone. Additional evidence that is inconsistent with the original mechanism was obtained using hole injection by a photoexcited anthraquinone in DNA. Instead of requiring the intermediacy of T center dot+, the strand damage patterns observed in those studies, in which thymidine is oxidized, are reproduced by independent generation of 2'-deoxyadenosin-N6-yl radical (dA(center dot)). Tandem lesion formation by dA(center dot) provides the basis for an alternative mechanism for thymidine oxidation ascribed to hole migration in poly(dA-T) sequences. Overall, these experiments indicate that the final products formed following DNA hole transfer in poly(dA-T) sequences do not result from deprotonation or hydration of T center dot+, but rather from deprotonation of the more stable dA(center dot+), to form dA(center dot), which produces tandem lesions in which 5'-flanking thymidines are oxidized.