N-Nitrosoindoles can efficiently transfer the nitroso group to nucleophilic targets in isolated purine nucleotides, causing depurination, deamination, and the formation of a novel guanine analogue, oxanine [Lucas, L. T., Gatehouse, D., and Shuker, D. E. G. (1999) J. Biol. Chem. 274, 18319-18326]. To determine the likely biological relevance of these modification pathways, the reactivity of 1-nitrosoindole-3-acetonitrile (NIAN), a model 3-substituted N-nitrosoindole, with oligonucleotides and calf thymus DNA was examined at physiological pH and temperature. Reaction of NIAN with single-stranded oligonucleotides containing various guanine motifs resulted in the production of single-strand break products at guanine sites due to the formation of alkali-labile lesions. The number of lesions increased with NIAN concentration and incubation time. Modification of calf thymus DNA by NIAN resulted in depurination, which gave the corresponding purine bases, deamination coupled with depurination, which gave xanthine, and the formation of oxanine. The former pathway was clearly the most important, and all reaction products exhibited a dose-response relationship. Cytosine and thymine residues were inactive toward NIAN. Further studies revealed an additional product in NIAN-treated duplex DNA containing a CCGG motif that was characterized as an interstrand cross-link, the yield of which increased with increasing NIAN concentration. These results indicate that the transnitrosating ability of NIAN to modify purine residues is preserved at the macromolecular level, with guanine residues appearing to be a primary site of reaction. All of these modification processes are potentially mutagenic events if they occur in vivo.