A recent analysis of data from nine studies provided convincing evidence that plasma estradiol measurements predict the risk of breast cancer in normal postmenopausal women. However, the median values detected by the various assays used in this study varied by 5-fold. These and other published data in normal postmenopausal women suggest that assays measuring low plasma estradiol concentrations suffer from problems of sensitivity, specificity, and precision. Availability of a practical, low-cost, specific, precise, and ultrasensitive estrogen assay might allow enhanced prediction of the risk of breast cancer and provide an objective means of selecting postmenopausal women for breast cancer prevention. A recombinant cell ultrasensitive bioassay (RCUB) for estrogen was recently validated for use in prepubertal children. We postulated that the RCUB might also prove useful for measurement of postmenopausal levels and designed the present study to examine this possibility. Thirty normal postmenopausal volunteers provided blood samples for measurement of estrogen by RCUB and, for comparison, by RIA. The estrogenic activity measured by RCUB [mean +/- sd, 11.9 +/- 10.9 pmol/liter (SI units, 3.23 +/- 2.96 pg/ml] was significantly lower than estradiol levels measured by RIA [43.7 +/- 44.0 pmol/liter (11.9 +/- 12.0 pg/ml)] in our volunteer subjects (P < 0.00001). Nonetheless, plasma estradiol levels measured by bioassay were significantly correlated with the estrogenic activity measured by RIA (r = 0.84) and by gas chromatography/tandem mass spectrometry (r = 0.85). To obtain biological evidence of the validity of the RCUB, we related plasma estrogen levels to body weight and body mass index and found highly significant correlations (r = 0.54 and r = 0.53, respectively). Surprisingly, 28 of 30 postmenopausal women were found to have estrogen levels in the prepubertal range with the RCUB. The levels detected by RCUB were similar to those previously reported using an ultrasensitive but less practical yeast bioassay. These results provide validation for the RCUB in postmenopausal women and suggest that it might prove useful for selection of women for drug therapy to prevent breast cancer.