A human specific monoclonal antibody (D5) raised against a Mr 36,000 cytosolic estrogen receptor component (RE) partially purified from human myometrium was used to develop a simple, rapid, and sensitive solid-phase immunoradiometric assay (IRMA) for the reactive antigen in tissue cytosols from breast tumors, myometrium, endometrium, and endometrial carcinomas. The IRMA did not detect antigen in RE-negative cytosols from human breast tumors and endometrial carcinomas. RE-positive cytosols from chick oviduct and calf and rat uteri failed to produce an IRMA response. A pilot study indicated a significant correlation (P less than 0.001) between D5 IRMA value and RE sites in breast tumors assayed by [3H]estradiol binding sites. The presence of D5 antigen was dependent on the presence of cytosolic RE but not progesterone receptor. RE-positive patients age 50 years and over demonstrated significantly higher D5 assay values than did patients under 50 years. The data suggest that the D5 antigen is a component of the estrogen receptor or coordinately regulated with the receptor in human cells and that the assay method may have clinical use.