A positive association between postmenopausal serum levels of total estradiol, percentage of free estradiol, and percentage of estradiol not bound to sex hormone-binding globulin (SHBG) and breast cancer risk was recently reported by the New York University Women's Health Study (P. Toniolo et al., J. Natl. Cancer Inst., 87: 190-197, 1995). Data from this prospective study are used to assess whether the observed associations differ according to estrogen receptor (ER) status of the tumor. Between 1985 and 1991, 7063 postmenopausal women donated blood and completed questionnaires at a large breast cancer screening clinic in New York City. Before 1991, 130 cases of first primary breast cancer were identified by active follow-up of the cohort. For each case, two controls were selected, matching the case on age at first blood donation and length of storage of specimens. Biochemical analyses were performed on sera that had been stored at -80 degrees since sampling. ER information was abstracted from pathology reports. Separate statistical analyses were conducted of ER-positive, ER-negative, and ER-unknown groups (53, 23, and 54 matched sets, respectively). In each of the 3 groups, the mean estradiol and the mean percentage of free estradiol were greater (21-28% and 6-7%, respectively) in cases than in controls. Conversely, the mean percentage of estradiol bound to SHBG was 9-12% lower in cases than in controls. The logistic regression coefficients measuring the strength of the association between estradiol and its free and SHBG-bound fractions and breast cancer risk were similar in the ER-positive, ER-negative, and ER-unknown groups. These data suggest that in postmenopausal women, the association of endogenous estrogens with breast cancer risk is independent of the ER status of the tumor. This result is more compatible with the hypothesis of a progression from ER-positive to ER negative tumors than with the hypothesis that ER status identifies two distinct types of breast cancer.