Endothelin (ET) potently inhibits arginine vasopressin (AVP)-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation and Na-K-adenosinetriphosphatase (Na-K-ATPase) activity in the inner medullary collecting duct (IMCD). At least two types of ET receptors exist: ETA [binds ET-1 > ET-3 = sarafotoxin S6c (S6c)] and ETB (binds ET-1 = ET-3 = S6c). We examined which of these receptors mediates biological actions of ET in freshly isolated rat IMCD cells. Binding studies revealed comparable displacement of 125I-ET-3 by ET-1, ET-3, and S6c, whereas 125I-ET-1 was displaced by ET-1 >> ET-3 = S6c. Together, these studies confirm the presence of receptors in the IMCD with ETA and ETB binding characteristics. ET-1, ET-3, and S6c were equipotent in reducing AVP-stimulated cAMP accumulation. BQ-123, at concentrations selective for ETA receptor antagonism, did not alter the effect of ET-1, ET-3, or S6c. Pertussis toxin or protein kinase C blockade, but not indomethacin, inhibited the effect of ET-1 and S6c on AVP-stimulated cAMP accumulation, consistent with activation of the same signal transduction pathways. ET-1 and S6c were equipotent in reducing forskolin-stimulated cAMP accumulation, ruling out inhibition of AVP-receptor interaction as a common mechanism of action. Finally, ET-1, ET-3, and S6c caused comparable stimulation of prostaglandin E2 (PGE2) accumulation, an effect that was not blocked by BQ-123. These data indicate that an ETB-like receptor mediates ET stimulation of PGE2 and inhibition of AVP-enhanced cAMP accumulation in the IMCD. The function of the ETA-like receptor in the IMCD remains to be determined.