AbstractRadioligand binding has been used for many years to identify new binding sites, characterize receptors, and identify novel ligands. Although various techniques have been developed to improve the efficiency of preparing the biological source of the receptors and for detecting bound radioligand, the principles of the assays remain the same. This unit reviews theory and provides examples of the parameters that can be calculated from radioligand binding data to characterize ligand‐receptor interactions. The important aspects of assay development and validation that allow meaningful interpretation are discussed. The selection of a radioligand, buffer and other assay components is critical to developing a useful binding assay. The nature of the binding interaction can also be probed by varying assay conditions.