The mechanism of UV-induced cleavage of duplex DNA containing 5-bromodeoxyuridine under anaerobic conditions was investigated via product analysis and kinetic isotope effect measurements using gel electrophoresis. KIEs strongly suggest that strand breaks result from abstraction of the C2‘ hydrogen atom from the 5‘-adjacent nucleotide by deoxyuridin-5-yl (1). It is proposed that under anaerobic conditions the C2‘ radical is subsequently oxidized by the cation radical of adenine. The resulting carbocation undergoes 1,2-hydride migrations from C1‘ and C3‘ competitively, giving rise to cleavage products containing 3‘-phosphate and the novel, labile 3‘-ketone (4). The identity of the latter product is supported by electrospray mass spectrometric analysis of the crude photolysate. The results also suggest that the formation of alkaline labile lesions proceed via hydrogen atom abstraction from the C2‘ position by 1.