The activation of signal transduction pathways by endothelin-1 or endothelin-3 were investigated in rat cerebromicrovascular endothelial cells. Endothelin-1 induced a rapid increase in inositol triphosphate (IP 3 ) formation in these cells, whereas endothelin-3 was only moderately effective at high concentrations. Both endothelins also increased uptake of 4 5 Ca 2 + in these cells. Endothelin-1-induced IP 3 formation or 4 5 Ca 2 + uptake were inhibited by endothelin ET A receptor antagonist BQ-123. Ryanodine, an inhibitor of intracellular Ca 2 + mobilization, selectively attenuated endothelin-1-induced 4 5 Ca 2 + uptake, whereas nickel or suramin inhibited endothelin-3-induced 4 5 Ca 2 + uptake. The results indicate that endothelin-1 elevates 4 5 Ca 2 + uptake in rat brain endothelial cells by mechanisms coupled to the mobilization of intracellular Ca 2 + stores. Both endothelin-1- and endothelin-3-induced 4 5 Ca 2 + uptake were inhibited by receptor operated Ca 2 + channel blocker SK & F 96365, whereas they were insensitive to dihydropyridine derivatives nifedipine and nitrendipine. The release of arachidonic acid from rat brain endothelial cells observed in response to endothelin-1 was inhibited by ryanodine or SK & F 96365, implicating participation of both intra- and extra-cellular components of Ca 2 + signaling in activating endothelial secretion of vasoactive substances.