Summary: The endothelin (ET) receptors on human placental membranes (HPMs), coupled to fluomicrospheres, have been characterized by examining the binding of 125I-ET-1 in a scintillation proximity assay (SPA). Specific binding of 125I-ET-1 was potently inhibited by ET-1 (IC50: 80 pM), ET-3 (IC50: 170 pM), and the ETB receptor-selective agonists sarafotoxin S6c (S6c; IC50: 210 pM) and alanine1,3,11,15-ET-1 (4-Ala-ET-1; IC50: 3.56 nM). In contrast, the ETA receptor-selective antagonist BQ123 (D-Val-Leu-D-Trp-D-Asp-Pro) only weakly (28% at 10 μM) inhibited 125I-ET-1 binding. In addition, the inhibition curves for ET-3 and 4-Ala-ET-1 were shallow, with slopes less than unity, indicating binding-site heterogeneity. In keeping with this finding, the presence of a small popula?? tion of ETA receptors was confirmed by the ability?? BQ123 (1 μM) to reduce the maximum binding capaci?? of the HPMs for 125I-ET-1 by ∽17%, without affecting the?? affinity for the radioligand. In conclusion, these resul?? suggest that the HPM-SPA system contains predom?? nantly (∽80%) ETB receptors, with a small ETA recepton?? population. These findings should be taken into accou?? when this assay system is used to identify novel endothe?? lin receptor ligands.