Treatment of B-form DNA with the antitumor antibiotic bleomycin in the presence of Fe2+ and O-2 affords both DNA strand scission and the formation of alkali-labile lesions, the proportion of which is quite sensitive to the concentration of O-2 present. The alkali-labile lesions can undergo fragmentation cleanly in the presence of n-butylamine to afford DNA fragments containing 5'- and 3'-phosphate termini at the site of the alkali-labile lesion. The mechanism of decomposition of the alkali-labile lesion was studied, leading to identification of a putative intermediate that is converted readily to an (oligo)nucleotide 3'-phosphate in the presence of n-BuNH2, as well as the identification of the byproduct of the fragmentation reaction containing the carbon atoms originally present within the alkali-labile lesion.