This study demonstrates the presence of a novel endothelin (ET) receptor subtype that displays high affinity for both ETA- and ETB-selective ligands. This subtype has been identified in canine spleen membranes using ETB-selective agonists ET-3, IRL-1620, sarafotoxin 6c (S6c) as well as ETA-selective antagonists BQ123 and related cyclic pentapeptides. Binding of 125I-ET-3 to canine spleen membranes was specific and saturable with an apparent dissociation constant of 130 pM and maximum binding (Bmax) of 240.0 fmol/mg protein. Although the apparent affinities obtained with 125I-ET-1 and 125I-ET-3 were comparable (90 and 130 pM, respectively), the maximum binding obtained with 125I-ET-3 was approximately 35% of that obtained with 125I-ET-1, which indicates that canine spleen possesses both ETA and ETB receptors in the ratio 65:35. Competition binding experiments using 125I-ET-3 and unlabeled ET-1, ET-3, S6c, and IRL-1620 suggested that although ET-1 and ET-3 displayed similar high affinity, S6c and IRL-1620 were 20-300-fold weaker than ET-1 and ET-3 in competing for 125I-ET-3 binding to canine spleen membranes. In addition, BQ123, an ETA-selective antagonist, displaced 125I-ET-3 binding from canine spleen with an IC50 value of 30 nM. Similar profiles were obtained with related cyclic pentapeptides. Electrophysiological studies performed on Xenopus laevis oocytes injected with canine spleen poly(A)+ RNA indicated that the ETB receptor present in these tissues is functional and displays the same pharmacology as that observed in binding studies using these membranes. As a comparison, both binding and functional studies were performed in canine lung and the data indicate that the ETB receptor present in this tissue is similar to that of the cloned human ETB receptor but different from that present in canine spleen. These observations were further confirmed by performing cross-linking experiments on these membranes. Although canine lung and cloned human ETB receptors displayed the same molecular weight bands with similar pharmacology, canine spleen ETB receptors displayed different molecular weight bands and different pharmacology. In addition, the ETB receptors present in canine spleen were also identified in canine bladder, monkey spleen and human spleen. Thus, the data presented in this manuscript provide evidence for the presence of a novel ETB receptor in different tissues as well as different species including human.