The present study aimed to characterize comparatively endothelin-1 (ET-1) receptors in rat tissues by radioligand binding assay using [¹²⁵I]ET-1 and to examine receptor binding after oral administration of bosentan. Significant amount of specific [¹²⁵I]ET-1 binding was detected in the lung, heart, kidney, bladder and cerebral cortex of rats. ET-1, bosentan, ambrisentan, and CI-1020 inhibited specific [¹²⁵I]ET-1 binding in these tissues in a concentration-dependent manner. The Hill coefficients of each agent in the rat lung and cerebral cortex and those of bosentan and ET-1 in the heart, kidney and bladder were close to unity, while the Hill coefficients of ambrisentan and CI-1020 in the heart, kidney and bladder were less than one. The nonlinear least squares regression analysis revealed the presence of high- and low-affinity ET-1 receptor sites in these tissues for ambrisentan and CI-1020. Oral administration of bosentan caused a dose-dependent decrease in specific [¹²⁵I]ET-1 binding in the rat lung, kidney and bladder, suggesting significant binding of the tissue ET-1 receptors in vivo . In conclusion, it has been shown that a significant amount of pharmacologically relevant ET-1 receptors may exist in rat tissues and that ET-1 receptor antagonists such as bosentan at pharmacological doses may exert some pharmacological effects by binding these ET-1 receptors.