A diet high in linoleic acid (an ?-6 PUFA) increased the formation of miscoding etheno (e) - DNA adducts in WBC-DNA of women, but not in men (Nair et al., Cancer Epidemiol Biomark Prev 1997;6:597-601). This gender specificity could result from an interaction between ?-6 PUFA intake and estrogen catabolism, via redox-cycling of 4-hydroxyestradiol (4-OH-E2) and subsequent lipid peroxidation (LPO). In this study, we investigated whether in premenopausal women LPO-derived adducts in WBC-DNA are affected by serum concentration of 2- and 4-hydroxyestradiol metabolites and by fatty acid intake. DNA extracted from buffy coat and plasma samples, both blindly coded from healthy women (N = 124, median age 40 year) participating in the EPIC-Heidelberg cohort study were analyzed. Three LPO-derived exocyclic DNA adducts, edA, edC and M1dG were quantified by immuno-enriched 32P-postlabelling and estradiol metabolites by ultra-sensitive GC-mass spectrometry. Mean M1dG levels in WBC-DNA were distinctly higher than those of edA and edC, and all were positively and significantly interrelated. Serum levels of 4-OH-E2, but not of 2-OH-E2, metabolites were positively related to etheno DNA adduct formation. Positive correlations existed between M1dG levels and linoleic acid intake or the ratios of dietary linoleic acid/oleic acid and PUFA/MUFA. Aerobic incubation of 4-OH-E2, arachidonic acid and calf thymus DNA yielded two to threefold higher etheno DNA adduct levels when compared with assays containing 2-OH-E2 instead. In conclusion, this study is the first to compare M1dG and etheno-DNA adducts and serum estradiol metabolites in human samples in the same subjects. Our results support a novel mechanistic link between estradiol catabolism, dietary ?-6 fatty acid intake and LPO-induced DNA damage supported by an in vitro model. Similar studies in human breast epithelial tissue and on amplification of DNA-damage in breast cancer patients are in progress.