[reaction: see text] An efficient method for the site-specific generation of 2-deoxyribonolactone oxidative DNA damage lesions from a "photocaged" nucleoside analogue was developed. A nucleoside phosphoramidite bearing a C-1' nitrobenzyl cyanohydrin was prepared and incorporated into DNA oligonucleotides using automated DNA synthesis. The caged analogue, which was stable in aqueous solution, was converted to the 2-deoxyribonolactone lesion by UV irradiation. DNA containing the caged analogue and the deoxyribonolactone site were characterized by electrospray mass spectrometry (ES-MS).