Newly discovered human DNA polymerase (pol) eta and kappa are highly expressed in the reproductive organs, such as testis, ovary, and uterus, where steroid hormones are produced. Because treatment with estrogen increases the risk of developing breast, ovary, and endometrial cancers, miscoding events occurring at model estrogen-derived DNA adducts were explored using pol eta and a truncated form of human pol kappa (pol kappaDeltaC). These enzymes bypassed N(2)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyguanosine (dG-N(2)-3MeE) and N(6)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyadenosine (dA-N(6)-3MeE), which were embedded in site-specifically modified oligodeoxynucleotide templates. Quantitative analysis of base substitutions and deletions occurring at the lesion site showed that pol kappaDeltaC was more efficient at incorporating dCMP opposite the dG-N(2)-3MeE lesion than pol eta. Surprisingly, the frequency of translesion synthesis beyond the dC*dG-N(2)-3MeE pair was 13% of the normal dC*dG pair and was 4 and 6 orders of magnitude higher than that of dC*(+)-trans-dG-N(2)-benzo[a]pyrene and dC*dG-C8-acetylaminofluorene pairs, respectively, suggesting that dG-N(2)-3MeE is a natural substrate for pol kappa. In contrast, the bypass frequency beyond the dT*dA-N(6)-3MeE pair was 7 orders of magnitude less than that for the normal dT*dA pair. dA-N(6)-3MeE is a more miscoding lesion than dG-N(2)-3MeE. Pol eta promoted incorporation of dAMP and dCMP at the dA-N(6)-3MeE lesion, while with pol kappaDeltaC, deletions were more frequently observed, along with incorporation of dAMP and dCMP opposite the lesion. These observations were also supported by steady-state kinetic studies. When taken together, the properties of pol eta and kappa are consistent with the mutagenic events attributed to estrogen-derived DNA adducts.