Exposure of aqueous solutions of DNA to X- or γ-rays, which induces the hydroxyl radical as one of the major reactive oxygen species (ROS), can result in the generation of a battery of single-nucleobase and bulky DNA lesions. These include the (5′R) and (5′S) diastereomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′- deoxyguanosine (cdG), which were also found to be present at appreciable levels in DNA isolated from mammalian cells and tissues. However, it remains unexplored how efficiently the cdA and cdG can be induced by Fenton-type reagents. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) with the use of the isotope-dilution technique, here we demonstrated that treatment of calf thymus DNA with Cu(II) or Fe(II), together with H2O2 and ascorbate, could lead to dose-responsive formation of both the (5′R) and (5′S) diastereomers of cdA and cdG, though the yields of cdG were 2-4 orders of magnitude lower than that of 8-oxo-7,8-dihydro-2′- deoxyguanosine. This result suggests that the Fenton reaction may constitute an important endogenous source for the formation of the cdA and cdG. Additionally, the (5′R) diastereomers of cdA and cdG were induced at markedly higher levels than the (5′S) counterparts. This latter finding, in conjunction with the previous observations of similar or greater levels of the (5′S) than (5′R) diastereomers of the two lesions in mammalian tissues, furnishes an additional line of evidence to support the more efficient repair of the (5′R) diastereomers of the purine cyclonucleosides in mammalian cells.