Cytoplasm from human mammary carcinoma contains an estradiol binding protein (EBP) with properties similar to if not identical with EBP found in rat mammary carcinoma. Human EBP is excluded on G100 Sephadex chromatography, precipitates with protamine sulfate, and sediments primarily at 8S in low ionic sucrose gradients. Increasing the ionic strength of the sucrose gradient or adding polyanions to the cytoplasm progressively reduces the sedimentation value to 4S. The affinity of EBP for 17β-estradiol is extremely high (Kd ∽ 5 × 10−M). There are structural restrictions for ligand binding since EBP does not recognize the common steroids hydrocortisone, progesterone, or testosterone.