The existence of estrogen receptors in the human prostate has long been a controversial issue. This may be explained partly by the apparent heterogeneity of estrogen-binding sites in prostatic tissue. We herein report on multiple binding sites for estrogens in cytosol as well as nuclear preparations of human prostatic tissues. One class of binding sites corresponds to the classical, high affinity estrogen receptor; the Kd for [3H]estradiol binding to the receptor was approximately 0.10 nM and the binding was specific for estrogens. The second class of binding sites appeared to have a Kd for [3H]estradiol in the range of 5-10 nM. This second, lower affinity class of binding sites markedly influenced studies of the classical receptor even at low ligand concentrations. Saturation analysis should be performed over a wide range of ligand concentrations (0.05-10 nM) to allow separation of the two binding components. Quantitation of estrogen receptor by a single point assay cannot be carried out accurately unless the low affinity binding component can be blocked. Multiple binding sites for estradiol were observed in the cytosol as well as in the nuclear salt extractable and salt-resistant compartments of normal, benign hyperplastic, and cancerous human prostates. Normal peripheral and cancerous prostates contained significantly (P less than 0.01) higher amounts of cytosol estrogen receptor compared to benign hyperplastic tissue.