The present study aimed to characterize bladder endothelin-1 (ET-1) receptor binding of clinically used ET-1 receptor antagonists by using [(125)I]ET-1. The inhibition of specific [(125)I]ET-1 binding was measured in the presence of ET-1 and its receptor antagonists. Specific binding of [(125)I]ET-1 in rat bladder was saturable and of high affinity, which characterized selective labeling of bladder ET-1 receptors. ET-1, bosentan, ambrisentan, and CI-1020 inhibited specific [(125)I]ET-1 binding in a concentration-dependent manner at nanomolar ranges of IC50. Nonlinear least squares regression analysis revealed the presence of high- and low-affinity ET-1 receptor sites for ambrisentan and CI-1020. Bosentan and ambrisentan significantly increased the dissociation constant for bladder [(125)I]ET-1 binding without affecting maximal number of binding sites (Bmax). Thus, bosentan and ambrisentan seem to bind to bladder ET-1 receptor in a competitive and reversible manner. Oral administration of bosentan caused a dose-dependent decrease in Bmax for bladder [(125)I]ET-1 binding, suggesting significant binding of bladder ET-1 receptors in vivo. A significant amount of pharmacologically relevant ET-1 receptors may exist in the bladder. These receptors may be implicated in the pathogenesis of lower urinary tract symptoms and may also be promising targets for the development of therapeutic agents.