Attempts at histochemical localization of estrogen receptor with anti-steroid antibody or some fluoresceinated estrogens have given unacceptable sensitivities and specificities when compared with biochemical methods or clinical response. In the present study a monoclonal antibody against estrogen receptor (H222 Sp gamma) was used on cryostat sections of freshly frozen breast tumors with a peroxidase-antiperoxidase immunoperoxidase technique. Biochemical receptor analyses were by dextran-coated charcoal analyses. Tumors from three separate cohorts of patients were studied as follows: population A, 62 primary breast cancers from 1983; population B, 72 primary lesions stored from 1976 to 1983; and population C, 23 patients with metastases, treated with hormonal therapy. Distinct staining was seen in the cell nucleus. A semiquantitative relationship was seen between histochemical score assessment of staining and biochemical assay in each cohort. The sensitivity and specificity using a threshold of 75 for the histochemical score and more than 20 femtomoles/mg of protein for dextran-coated charcoal analyses were as follows: population A, specificity, 89%, and sensitivity, 95%; population B, specificity, 94%, and sensitivity 88%; and for population C, the comparison was with objective clinical response yielding specificity, 89%, and sensitivity, 93%.