Design, methods, and study population of a long-term multidisciplinary investigation of benign and malignant breast disease were reported. This initial report focused on the relation of menstrual, reproductive, and other factors to serum and breast fluid estrogen measures [estradiol (E2), estrone (E1), percent free estrogen, and sex hormone binding globulin] among control women. After adjustment for the factors found to be related to the various estrogen measures, estrogen levels in women with benign and malignant disease were compared to those of controls. Findings were as follows: a) little evidence of any relation of most breast cancer risk factors with the various serum estrogen parameters studied; b) differences in breast fluid estrogen levels that may be relevant to the protective effect of parity on breast cancer risk; c) markedly higher levels of E2 and E1 in breast fluid than in serum and no evidence of a correlation of serum with breast fluid measures; d) no support for the hypothesis that breast cancer patients have higher serum percent free E2 than controls or women with benign breast disease; and e) higher breast fluid E2 and E1 levels in women with biopsied benign breast disease than in controls.