8,5'-Cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) are among the major lesions formed in DNA by hydroxyl radical attack on 2'-deoxyadenosine and 2'-deoxyguanosine, respectively, followed by intramolecular cyclization between C5' and C8. Mechanisms of formation of these unique tandem lesions were elucidated. The 8,5'-cyclization causes an unusual puckering of the sugar moiety giving rise to significant distortion in the DNA double helix. Methodologies were developed for the measurement of these lesions in DNA by mass spectrometry coupled either with gas chromatography or high performance liquid chromatography. Both techniques allowed identification and quantification of both R- and S-diastereomers of cdA and cdG in DNA in vitro and in vivo. Because of the 8,5'-covalent bond between the sugar and base moieties in the same nucleoside, cdA and cdG are repaired by nucleotide excision repair rather than by base excision repair. Thus, these lesions may play a role in diseases with defective nucleotide excision repair. Their biological effects include blocking DNA polymerases, inhibition of gene expression, transcriptional mutagenesis among others. Accumulation of cdA and cdG was observed in tissues in vivo in connection to disease and environmental conditions, suggesting an important role for these lesions in disease processes including carcinogenesis and neuronal death.