Reactive oxygen species (ROS) can be induced by both endogenous and exogenous processes, and they can damage biological molecules including nucleic acids. It was shown that X- or gamma-ray irradiation of aqueous solutions of DNA, during which *OH is one of the major ROS, can lead to the formation of intrastrand cross-link lesions where the neighboring nucleobases in the same DNA strand are covalently bonded. Previous 32P-postlabeling studies suggested that the intrastrand cross-link lesions may arise from Fenton reaction, which also induces the formation of *OH; the structures of the proposed intrastrand cross-link lesions, however, have not been determined. Here, we showed for the first time that the treatment of calf thymus DNA with Cu(II)/H2O2/ascorbate could lead to the formation of an intrastrand cross-link lesion, i.e., G wedge T, where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon. LC-MS/MS quantification results showed dose-responsive formation of G wedge T. In addition, the yield of the intrastrand cross-link was approximately 3 orders of magnitude lower than those of commonly observed single-base lesions, that is, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-formyl-2'-deoxyuridine. The induction of intrastrand cross-link lesion in calf thymus DNA by Fenton reagents in vitro suggests that this type of lesion might be formed endogenously in mammalian cells.