The concentration of estrogen receptor protein in breast-tumor tissue is generally expressed in units of femtomoles of estradiol bound by the receptor per milligram of cytosol protein. The sensitivity of the estrogen receptor radioligand assay is therefore related to the specific activity of the steroid label used for the binding assay, the amount of the receptor protein in the volume of cytosol used, and the protein concentration in the cytosol. In this paper, we discuss factors affecting the sensitivity of the estrogen receptor assay and present various approaches for optimizing the assay. We also describe a procedure that involves a radioiodinated estradiol of high specific activity, a micro-technique for preparing tumor cytosol, and a micro-assay procedure with which the estrogen receptor protein can be measured in as little as 50 mg (wet weight) of tissue.