A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeuAspIleIleTrp (BQ-3020), is highly selective for ET B receptors. BQ-3020 displaces [ 125I]ET-1 binding to ET B receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC 50: 0.2nM) at a concentration 4,700 times lower than that to ET A receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC 50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ET A antagonist BQ-123 failed to inhibit this BQ-3020-induced vasocontraction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ET B agonistic activity. The radioligand [ 125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd= 34.4Pm), where it scarcely binds to VSMC. [ 125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC 50: 0.07–0.17nM). However, the binding of [ 125I]BQ-3020 was insensitive to the ET A antagonist BQ-123 and other bioactive peptides. Both [ 125I]ET-1 and [ 125I]BQ-3020 show slow onset and offset binding kinetics to ET B receptors. These data indicate that the radioligand [ 125I]BQ-3020 selectively labels ET B receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu 6 to Trp 21, but not on the structure formed by its two disulfide bridges.