Endothelin-1 (ET-1) acutely increases Na/H antiporter activity in OKPET(B)6 cells, an opossum kidney proximal tubule cell line transfected with ETB receptor cDNA. The purpose of the present study was to examine the chronic effect of ET-1 on Na/H antiporter activity in OKP cells and to examine whether Na/H exchanger (NHE)-3 mRNA and protein abundance are regulated by ET-1, Quiescent OKPET(B)6 cells were treated with 10 nM ET-1 for 3, 6 or 24 h and Na/H antiporter activity was assayed. The Na/H antiporter activity in 3-h ET-1-treated cells was not different from controls. However, Na/H antiporter activity was significantly decreased by 29% at 6 h and 72% at 24 h. The effect of ET-1 on Na/H antiporter activity was blocked by BQ788, an ETB receptor antagonist, but not BQ123, an ETA receptor antagonist, The NHE-3 mRNA abundance in ET-1-treated cells was not different from controls at 3 h, However, there was a significant decrease in NHE-3 mRNA abundance at 6 and 24 h, There was also a significant decrease in NHE-3 protein abundance at 6 and 24 h. In summary, ET-1 chronically inhibits NHE-3 in OKPET(B)6 cells.