Estrogen receptors (ER) are detected in 50–85% of all breast tumors, and are clinically important because they tend to identify patients with a higher probability of responding to hormonal or endocrine manipulations. However, ∼30–40% of all ER+patients do not respond to hormonal manipulations. The lack of response to hormonal manipulations in ER+patients could be the result of nonfunctional ER, as determined by its inability to recognize and bind to specific DNA-responsive elements and/or its inability to recruit other transcriptional activation factors. The functional status of ER in 34 human breast tumors was assessed by determining the structural integrity of the ER DNA-binding domain using site-directed monoclonal anti-estrogen receptor antibody and sucrose density gradient analysis. Based on the fraction of ER containing an intact DNA-binding domain, the tumors were classified into three groups: group I with >65% of intact ER, group II with >30% of intact ER. and group III with <30% of intact ER. Clinical and pathologic data were obtained only for patients who were treated with the anti-estrogen tamoxifen and correlated with ER functional status. In group I, 11 of 13 (84.6%) patients were responsive to hormonal therapy with favorable clinical outcome; two patients had unfavorable clinical outcome. In group II, 13 of 15 patients (86.7%) had favorable clinical outcome and two patients (13.3%) had unfavorable outcome. In group III, three of six patients appeared to be hormone responsive with favorable clinical outcome, and three of the patients in this group had unfavorable response to therapy. ER functional status may be linked to hormone responsiveness in breast cancer and the resistance of some ER+tumors to anti-estrogen treatment may be explained, in part, by dysfunctional ER. This approach may permit the development of new clinical assays based on ER functional status, which could facilitate selection of patients who are likely to benefit from anti-estrogen therapy.