Nitrogen-centered nucleoside radicals are cornmonly produced reactive intermediates in DNA exposed to gamma-radiolysis and oxidants, but their reactivity is not well understood. Examination of the reactivity of independently generated 2'deoxyadenosin-N6-yl radical (dA.) reveals that it is an initiator of tandem lesions, an important form of DNA damage that is a hallmark of y-radiolysis. dA. yields O-2 -dependent tandem lesions by abstracting a hydrogen atom from the CS-methyl group of a 5'-adjacent thymidine to form 5-(2'-deoxyuridinyOmethyl radical (T.). The subsequently formed thymidine peroxyl radical adds to the S'-adjacent dG, ultimately producing a S'-OxodGuo-fdU tandem lesion. Importantly, the initial hydrogen abstraction repairs dAo to form dA. Thus, the involvement of dAo in tandem lesion formation is traceless by product analysis. The tandem lesion structure, as well as the proposed mechanism, are supported by LC-MS/MS, isotopic labeling, chemical reactivity experiments, and independent generation of T.. Tandem lesion formation efficiency is dependent on the ease of ionization of the 5'-flanking sequence, and the yields are >27% in the 5'-d(GGGT) flanking sequence. The traceless involvement of dA. in tandem lesion formation may be general for nitrogen-centered radicals in nucleic acids, and presents a new pathway for forming a deleterious form of DNA damage.