This study investigates the presence and quantification of cytoplasmic estradiol-binding protein (EBP) in human breast cancer tissues. Using sucrose gradient centrifugation, EBP was found to sediment at 8S and 4S, with variable non-specific estradiol binding in the 4S region. Scatchard analysis revealed a dissociation constant of 2.6 × 10^-8 M for the estradiol-EBP interaction, indicating high affinity. Quantitation of EBP sites in 64 primary and metastatic breast tumors showed a wide range from 0 to 612 fmol per mg of cytoplasmic protein. Specific 8S binding was not detected in samples with less than 9.0 fmol EBP per mg cytoplasmic protein. The correlation between abundant tumor EBP and endocrine-induced breast cancer regressions suggests that precise quantitation of EBP may be a valuable prognostic indicator for endocrine therapy in metastatic breast cancer. The study emphasizes the need for quantitative assays and controls to ensure the specificity of EBP measurements.This study investigates the presence and quantification of cytoplasmic estradiol-binding protein (EBP) in human breast cancer tissues. Using sucrose gradient centrifugation, EBP was found to sediment at 8S and 4S, with variable non-specific estradiol binding in the 4S region. Scatchard analysis revealed a dissociation constant of 2.6 × 10^-8 M for the estradiol-EBP interaction, indicating high affinity. Quantitation of EBP sites in 64 primary and metastatic breast tumors showed a wide range from 0 to 612 fmol per mg of cytoplasmic protein. Specific 8S binding was not detected in samples with less than 9.0 fmol EBP per mg cytoplasmic protein. The correlation between abundant tumor EBP and endocrine-induced breast cancer regressions suggests that precise quantitation of EBP may be a valuable prognostic indicator for endocrine therapy in metastatic breast cancer. The study emphasizes the need for quantitative assays and controls to ensure the specificity of EBP measurements.