We previously suggested the presence of functionally atypical endothelin (ET) A receptors in the rabbit iris sphincter. Here, we further characterized the ET receptor by a radioligand-receptor binding study utilizing a membrane fraction of the rabbit iris. In addition, we functionally confirm the presence of an atypical ET(A) receptor in the iris dilator similar to that in the iris sphincter. In binding experiments, [125I]ET-1 was completely displaced by ET-3 in a biphasic fashion, but only partially by BQ-123 and ET(B) ligands. In the presence of RES-701, ET-3 and sarafotoxin (SRTX)-b completely displaced [125I]ET-1 in a monophasic fashion, but with shallow slopes. Moreover, ET-1, ET-3 and SRTX-b completely displaced [3H]BQ-123 with IC50 values of 0.8, 81 and 4.4 nM, respectively, but with slopes of ET-3 and SRTX-b being again shallow. In iris dilator muscles, ET-3 showed lower and SRTX-b showed higher contractile activities than ET-1. SRTX-c was inactive. BQ-123 more preferentially antagonized ET-3 and SRTX-b than ET-1, with the Schild plot slope of SRTX-b being shallow. Thus, functional experiments suggested the presence of atypical ET(A) receptors in the iris dilator similar to the iris sphincter. However, the binding experiments suggested the presence of rather typical ET(A)- and ET(B)-like receptors. Therefore, we apparently failed to show ET binding sites corresponding to functionally atypical ET(A) receptors.