To examine the mechanisms by which endothelin (ET) regulates the Na/H antiporter isoform, NHE-3, OKP cells were stably transfected with ET(A) and ET(B) receptor cDNA. In cells overexpressing ET(B), but not ET(A) receptors, ET-1 increased Na/H antiporter activity (JNa/H). This effect was inhibited by a nonselective endothelin receptor blocker and by a selective ET(B) receptor blocker but was not inhibited by an ET(A) selective receptor blocker. In ET(B)-overexpressing cells, 10(-8) M ET-1 inhibited adenylyl cyclase, but protein kinase A inhibition and pertussis toxin pretreatment did not affect Na/H antiporter activation by ET-1. ET-1 caused a transient increase in cell [Ca2+], followed by a sustained increase. Increases in cell [Ca2+] were partially inhibited by pertussis toxin. ET-1-induced increases in J(Na/H) were 50% inhibited by clamping cell [Ca2+] low with BAPTA, and by KN62, a Ca-calmodulin kinase inhibitor. Inhibitors of protein kinase C, cyclooxygenase, lipoxygenase, and cytochrome P450 and cyclic GMP were without effect. In ET(A)-overexpressing cells, ET-1 increased cell [Ca2+] but did not increase JNa/H. In summary, binding of ET-1 to ET(B) receptors increases Na/H antiporter activity in OKP cells, an effect mediated in part by increases in cell [Ca2+] and Ca-calmodulin kinase. Increases in cell [Ca2+] are not sufficient for Na/H antiporter activation.