Reactive oxygen species can give rise to a battery of DNA damage products including the 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) tandem lesions. The 8,5'-cyclopurine-2'-deoxynucleosides are quite stable lesions and are valid and reliable markers of oxidative DNA damage. However, it remains unclear how these lesions compromise DNA replication in mammalian cells. Previous in vitro biochemical assays have suggested a role for human polymerase (Pol) η in the insertion step of translesion synthesis (TLS) across the (5'S) diastereomers of cdA and cdG. Using in vitro steady-state kinetic assay, herein we showed that human Pol ι and a two-subunit yeast Pol ζ complex (REV3/REV7) could function efficiently in the insertion and extension steps, respectively, of TLS across S-cdA and S-cdG; human Pol κ and Pol η could also extend past these lesions, albeit much less efficiently. Results from a quantitative TLS assay showed that, in human cells, S-cdA and S-cdG inhibited strongly DNA replication and induced substantial frequencies of mutations at the lesion sites. Additionally, Pol η, Pol ι, and Pol ζ, but not Pol κ, had important roles in promoting replication through S-cdA and S-cdG in human cells. Based on these results, we propose a model for TLS across S-cdA and S-cdG in human cells, where Pol η and/or Pol ι carries out nucleotide insertion opposite the lesion, whereas Pol ζ executes the extension step.