The mechanism of oxidation of guanine residues on double-stranded oligonucleotides (ODNs) by the chemical nuclease Mn-TMPyP/KHSO5 is reported. By using HPLC coupled to an electrospray mass spectrometer (ESI/MS) the different oxidized ODN strands were directly analyzed, and labeling experiments in (H2O)-O-18 allowed us to propose a two-electron oxidation mechanism for guanine residues engaged in double-stranded DNA. We found that the imidazolone derivative (dIz) was formed by trapping of a guanine-cation by a water molecule. Two reaction intermediates on the pathway of the formation of dIz were observed: 5,8-dihydroxy-7,8-dihydroguanine and an oxidized guanidinohydantoin intermediate. Furthermore, a secondary route of guanine oxidation leading to parabanic acid was also evidenced. The mechanism of the different routes of guanine oxidation in double-stranded DNA has been discussed in detail.