This PerformanceBased Test Guideline PBTG describes in vitro assays, which provide the methodology for human recombinant in vitro assays to detect substances with estrogen receptor binding affinity hrER binding assays. It comprises two mechanistically and functionally similar test methods for the identification of estrogen receptor i.e. ERα binders and should facilitate the development of new similar or modified test methods. The two reference test methods that provide the basis for this PBTG are the FreybergerWilson FW In Vitro Estrogen Receptor ER Binding Assay Using a Full Length Human Recombinant ERα, and the Chemical Evaluation and Research Institute CERI In Vitro Estrogen Receptor Binding Assay Using a Human Recombinant Ligand Binding Domain Protein. This assay measures the ability of a radiolabeled ligand 3H17βestradiol to bind with the ER in the presence of increasing concentrations of a test chemical i.e. competitor. Test chemicals that possess a high affinity for the ER compete with the radiolabeled ligand at a lower concentration as compared with those chemicals with lower affinity for the receptor. This assay consists of two major components a saturation binding experiment to characterise receptorligand interaction parameters and document ER specificity, followed by a competitive binding experiment that characterises the competition between a test chemical and a radiolabeled ligand for binding to the ER. These test methods are being proposed for screening and prioritisation purposes, but also provide mechanistic information that can be used in a weight of evidence approach.