IRL1620, a specific endothelin ETB receptor agonist, induced relaxation followed by contraction in the guinea-pig ileum, as did endothelin-1. Both components of the response were concentration-dependent in the range studied. Repeated administration of IRL1620 induced tachyphylaxis only of the contractile component, whereas endothelin-1 desensitized both components. BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]), a specific endothelin ETA receptor antagonist, did not inhibit the relaxation induced by either agonist, although it did inhibit the contraction induced by endothelin-1, but not by IRL1620. PD145065 (Ac-(D-Bhg-Leu-Asp-Ile-Ile-Trp) (D-Bhg = 5H-dibenzyl[a,d]cycloheptene-10,11-dihydroglycine)), a combined endothelin ETA/endothelin ETB receptor antagonist, inhibited the contractile effects of both endothelin-1 and IRL1620 and also inhibited the relaxation induced by IRL1620. Apamin, a Ca(2+)-activated K+ channel blocker, inhibited only the endothelin-1-induced relaxation. Our studies suggest that two endothelin ETB receptor subtypes mediate relaxation in the guinea-pig ileum: one is less sensitive to PD145065 but apamin-inhibitable, and the other is more sensitive to PD145065 but not apamin-inhibitable. Our results also suggest that both endothelin ETA and endothelin ETB receptor subtypes mediate contraction in the ileum.