Endothelial cells from brain microvessels (BCEC) express high affinity receptor sites for endothelin-1 that recognize endothelin-3 with a low affinity (Vigne, P., Marsault, R., Breittmayer, J.P. & Frelin, C. (1990) Biochem. J. 266, 415-420). Binding experiments using 125I-endothelin-3 showed the presence in BCEC of a new class of receptor sites that had a high affinity for endothelin-3 (Kd = 0.8 nM), endothelin-1 (Kd = 0.8 nM), and sarafotoxin S6b (Kd = 0.3 nM). Endothelins activated phospholipase C in BCEC and produced transient increases in intracellular Ca2+ with properties of a low affinity endothelin-3 receptor. Endothelins also increased 22Na+ uptake via the Na+/H+ antiporter in BCEC. Concentrations for half-maximum activation (endothelin-1, 0.5 nM; sarafotoxin S6b, 1 nM; endothelin-3, 2 nM) were close to the Kd values determined in 125I-endothelin-3-binding experiments. The action of endothelins on Na+/H+ exchange was not mimicked by phorbol myristate acetate, it was not reversed by staurosporine, and it did not correlate with the phosphorylation of the 80-kDa protein. These results indicated that the action of endothelins on Na+/H+ exchange did not involve protein kinase C. It is concluded that BCEC coexpress two types of functional receptor sites for endothelins: (i) a high affinity endothelin-1, low affinity endothelin-3 receptor that is coupled to phospholipase C and to intracellular Ca2+ mobilization, and (ii) a high affinity endothelin-1, high affinity endothelin-3 receptor that controls Na+/H+ exchange activity via a protein kinase C-independent mechanism.