The biological activity of natural estrogens is influenced by the degree to which they bind to serum proteins. To determine directly how serum affected the uptake of estradiol, we compared the whole cell uptake of [3H]estradiol in intact MCF-7 human breast cancer cells from serum-free medium with the uptake from 100% serum from adult men. In estrogen receptor saturation assays, 28.9 times more estradiol was required in serum to occupy the same number of estrogen receptors as was required in serum-free medium (SFM), suggesting that the effective free fraction of estradiol in adult male serum was 3.46% (1/28.9). Since most xenoestrogens are not available in tritium-labeled form, the cell uptake of unlabeled xenoestrogens could not be measured directly with saturation analysis. Therefore, we developed the relative binding affinity-serum modified access (RBA-SMA) assay to determine the effect of serum on the access of nonradioactive xenoestrogens to estrogen receptors within intact MCF-7 cells. Serum modified access (SMA) was calculated by dividing the relative binding affinity (RBA, relative to estradiol) measured in 100% serum, by the RBA measured in serum-free medium. An SMA > 1 indicated that the xenoestrogen had greater access to estrogen receptors than estradiol from serum. In contrast, an SMA < 1 indicated that the xenoestrogen had less access to estrogen receptors from serum than did estradiol. The synthetic estrogen diethylstilbestrol (DES) binds poorly to sex hormone binding globulin (SHBG), and DES showed enhanced access in serum, SMA = 6.2. Additional calculations through the Ki (inhibition constant) indicated that this corresponded to an effective free fraction of 26.9% for DES in serum. The phytoestrogens, coumestrol, genistein, and equol, showed substantial enhanced access in serum, over 10-fold relative to estradiol (SMA = 12.1, 10.3, and 11.3, respectively), and effective free fractions in serum of 47.8, 45.8, and 49.7%, respectively. Since most in vitro assays of xenoestrogens do not address how serum influences their bioactivity, the estrogenic activity of these phytoestrogens would be underestimated. Conversely, biochanin A showed decreased access from serum (SMA = 0.44) and had an effective free fraction of 2.4%; its estrogenic activity would be overestimated in serum-free assays.