1 A specific and sensitive radioreceptor assay ahs been devised which is based on high affinity, saturable binding of 9 nM [3H]-ouabain to the total particulate fraction isolated from dog heart. Ouabain and other cardiac glycosides, including the aglycones, were about equipotent in their ability to displace [3H]-ouabain from its receptor, the IC50s ranging from 10 to 30 nM. 2 The only other substances found to compete significantly in the assay were derivatives of hydroxyprogesterone having a 17 alpha-acetate substituent: chlormadinone acetate, megestrol acetate, cyproterone acetate and medroxyprogesterone acetate, with IC50s of 2, 7.4, 9 and 21 microM, respectively. Prednisolone-3,20-bisguanyl-hydrazone, reported to have inotropic activity, gave an IC50 of 6.4 microM. Cyproterone-17 alpha-OH was less active (IC50 90 microM) than cyproterone-17 alpha-acetate. 3 A large number of peptide and protein hormones, steroid hormones and their metabolites, amines, and drugs were inactive.