An endothelin-1 (ET-1) receptor-binding assay, in microtiter format, has been developed for use in high throughput chemical or natural product screening. A rat smooth muscle, clonal cell line derived from embryonic thoracic aorta and designated A10 has been shown to consistently express high-affinity ET-1 receptors. A 96-well microtiter filtration plate, with individual PVDF membranes attached to the bottom of each well, was used for separation. In saturation binding assays, using [125I]Tyr13-ET-1, Scatchard analysis was monophasic, indicating a single high-affinity population of receptors with Kd = 0.12 nM with approximately 40,000 receptors per cell. The Ki values for peptides, known to bind at the ET receptor, were as follows: ET-1, 0.14 nM; ET-2, 0.16 nM; sarafotoxin S6b, 0.6 nM; VIC, 0.2 nM; ET-3, 16 nM; and big human ET, greater than 1 microM. ET receptors on A10 cells were stable for at least 2 months when stored at -20 degrees C. The assay is suitable for automation, because it is stable and reproducible. This method gave a 90% reduction in radioactive waste compared to tissue homogenate assays that use glass fiber filtration and cell harvesters.