Increased recognition of the importance of selective hormone binding in initiation of steroid action in target tissues led us to study estrogen binding by human breast cancer. Mastectomy specimens were obtained from 4 premenopausal and 11 post-menopausal women ranging in age from 37 to 83 yr. They were characterized histologically as infiltrating ductal carcinoma in 9, adenocarcinoma in 5, and colloid carcinoma in one. The capacity of the high-speed ultracentrifugal supernatant (cytosol) of homogenates of mammary carcinoma, glandular tissue and fat, was tested for the ability to bind tritiated 17β-estradiol (E2-3H) by the radioligand binding technique we previously described for uterus. Glandular tissue and fat from all 15 mastectomy specimens failed to bind E2-3H, as did 7 tumors. One tumor had slight steroid binding activity which was unsaturable and nonspecific. Seven tumor cytosols had E2 binding capacities in excess of 0.8, 0.8, 5.1, 10.0, 10.5, 19.5 and 21.3 × 10−14 moles/mg protein. Testosterone, cortisol and progesterone did not compete for E2 binding sites. Quantitative relative binding affinity (RBA) studies of the 5 most active cytosols showed that other estrogens were moderate to weak competitive inhibitors of E2-3H binding with epiestriol > estrone > estriol > 17α-estradiol. The RBA of 4 nonestroidal estrogen inhibitors were erythro-MEA >dimethylstilbestrol > cis clomiphene = CN-55-945-27. E2 levels in cytosols from 6 patients were low. E1 concentrations, on the other hand, ranged up to 65 and 46 pg/ml for 2 specimens from glandular tissue and from 20 to 60 pg/ml in 5 tumor cytosols, representing about 300 mg/supernatant protein. Particularly in post-menopausal women, E1 may be involved in estrogenic stimulation of the breast. It is tempting to suggest that specific binding to cytosol may be a prerequisite for hormonal stimulability of breast carcinoma.