Estrogen receptors in frozen section of the rat uterus were demonstrated by a radiolabeled ligand binding technique. The bound hormone was extracted with ethanol and measured by liquid scintillation. The binding of 3H-estradiol-17 beta at various molar concentrations was inhibited by a 100-fold excess of DES, and the bound 3H-estradiol resisted exhaustive washings at 4 degrees C for 16 h. These binding sites were not present in the sections of the spleen, and perhaps at a very low concentration in the myocardium. Thus their binding behavior and distribution pattern are consistent with those of specific estrogen receptors. The hydrophilic fluorescent estradiol conjugate, 17 beta-estradiol-6-CMO-BSA-FITC was found to be a highly effective competitor against binding of 3H-estradiol to its receptors in tissue sections, and is considered a useful histochemical reagent for localizing target cells with high concentrations of estrogen receptors. Estrogen receptor sites in frozen sections of human breast cancer were also measured by this radiolabeled ligand binding technique, and expressed in femtomoles of hormone bound per 1,000 cancer cells. The values were parallel to the histochemical findings in terms of percentage of the estrogen receptor-positive in the cancer cell population.