Of the four major nucleosides in DNA, 2′-deoxyguanosine (dG) is the most easily oxidized. The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) by oxidation of free dG, as well as of dG in DNA, by hydrogen peroxide (H2O2) and the reducing agent ascorbate (HAsc−) was measured using HPLC with high sensitivity electrochemical detection. High concentrations of the hydroxyl radical (OH˙) scavengers PBN, DMPO, pentoxifylline and imidazole had a very limited protective effect on 8-oxodG formation, which provides evidence against free OH˙ production as the major mechanism. A possible mechanism involves a “two-electron reduction of H2O2” with oxidation of HAsc− and dG, producing the guanine radical cation (dG˙+) leading to 8-oxodG. This mechanism is, based on changes in Gibbs free energy (ΔG0′) at pH 7, much more favourable than OH˙ production. Catalase from Aspergillus niger and the aminoxyl TEMPO almost completely inhibited 8-oxodG formation, whereas bovine catalase cleaved the free dG molecule. Substantial 8-oxodG formation from free dG occurred even at 0 °C.