Estrogen receptors (ERs) in human breast cancer were studied using specific quantitative techniques to measure cytoplasmic estradiol-binding protein (EBP). Sucrose gradient centrifugation revealed EBP sedimenting at 8S and 4S. The 4S region showed variable nonspecific estradiol binding, requiring controls to ensure specificity. Scatchard analysis found a dissociation constant of approximately 2.6×10⁻¹⁰ M, indicating high affinity of estradiol for EBP. Quantitation of EBP sites in 64 breast tumors showed a continuous range from 0 to 612 fmol per mg of cytoplasmic protein. Specific 8S binding was not detected in specimens with less than 9.0 fmol EBP per mg protein. The presence of abundant EBP correlated with endocrine therapy responses in breast cancer. This suggests that precise quantitation of EBP in primary tumors could predict endocrine therapy outcomes in metastatic breast cancer. The study used sucrose gradient centrifugation and DCC methods to quantify EBP. The DCC method effectively removed non-bound estradiol, allowing measurement of bound estradiol. Scatchard analysis confirmed the specificity of EBP-estradiol interactions, with a dissociation constant of 2.6×10⁻¹⁹ M. The study found that EBP concentrations varied widely in human breast tumors, with an 8-10S peak indicating EBP presence. Nonspecific binding was a concern, necessitating controls. The DCC method provided accurate quantitative data, while sucrose gradient centrifugation offered specificity proof. The 4-5S peak could represent nonspecific binding, requiring separate analysis. The study also noted that endogenous estradiol levels could influence EBP measurements. In conclusion, specific quantitative assays for EBP in human breast cancer are now available. These assays correlate with clinical responses and enhance understanding of endocrine therapy in breast cancer. The study highlights the importance of accurate EBP quantitation for predicting treatment outcomes.Estrogen receptors (ERs) in human breast cancer were studied using specific quantitative techniques to measure cytoplasmic estradiol-binding protein (EBP). Sucrose gradient centrifugation revealed EBP sedimenting at 8S and 4S. The 4S region showed variable nonspecific estradiol binding, requiring controls to ensure specificity. Scatchard analysis found a dissociation constant of approximately 2.6×10⁻¹⁰ M, indicating high affinity of estradiol for EBP. Quantitation of EBP sites in 64 breast tumors showed a continuous range from 0 to 612 fmol per mg of cytoplasmic protein. Specific 8S binding was not detected in specimens with less than 9.0 fmol EBP per mg protein. The presence of abundant EBP correlated with endocrine therapy responses in breast cancer. This suggests that precise quantitation of EBP in primary tumors could predict endocrine therapy outcomes in metastatic breast cancer. The study used sucrose gradient centrifugation and DCC methods to quantify EBP. The DCC method effectively removed non-bound estradiol, allowing measurement of bound estradiol. Scatchard analysis confirmed the specificity of EBP-estradiol interactions, with a dissociation constant of 2.6×10⁻¹⁹ M. The study found that EBP concentrations varied widely in human breast tumors, with an 8-10S peak indicating EBP presence. Nonspecific binding was a concern, necessitating controls. The DCC method provided accurate quantitative data, while sucrose gradient centrifugation offered specificity proof. The 4-5S peak could represent nonspecific binding, requiring separate analysis. The study also noted that endogenous estradiol levels could influence EBP measurements. In conclusion, specific quantitative assays for EBP in human breast cancer are now available. These assays correlate with clinical responses and enhance understanding of endocrine therapy in breast cancer. The study highlights the importance of accurate EBP quantitation for predicting treatment outcomes.