Endothelin-1 (ET-1) induces contraction of vascular smooth muscle through binding to endothelin type A receptor (ET(A)R). COS-7 cells stably expressing high levels of the ET(A)R were established (designated COS-7(ET(A)R)). The COS-7(ET(A)R) cell bound [(125)I]ET-1 with a K(d) of 932+/-161 pM and a B(max) of 74+/-13 fmol/2x10(5) cells. [(125)I]ET-1 binding was inhibited by ET-1 and the ET(A)R antagonist BQ-610, but not by the endothelin type B receptor (ET(B)R) antagonist BQ-788. In clones expressing two ET(A)R mutants containing D46N or R53Q substitutions in the first extracellular domain of the receptor, [(125)I]ET-1 binding activity was dramatically reduced. This suggests that these single amino acid substitutions alter the three-dimensional structure of the ligand-binding domain of the ET(A)R. Using COS-7(ET(A)R) cell, we showed that Ca(2+) or Mg(2+) was essential for ET-1 binding to the ET(A)R and that ET-1 treatment induced postreceptor signaling, that is, intracellular accumulation of cyclic AMP (cAMP) and Ca(2+) mobilization. The COS-7(ET(A)R) established in this study will be a useful tool for screening ET-1 antagonists for treating hypertension.