We investigated the role and mechanism of hydrogen peroxide (H₂O₂) in regulation of NaCl transport in primary inner medullary collecting duct (IMCD) cells. IMCD cells were isolated from wild type mice and grown onto semipermeable membranes, and short-circuit current (Isc) was determined by Ussing chamber. Exposure of IMCD cells to H₂O₂ at a range of 100-300 µM caused a rapid increase in Isc in a time- and dose-dependent manner. This increase was almost abolished by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel inhibitors, diphenylamine-2-carboxylic acid (DPC) and CFTR inhibitor-172. In contrast, the magnitude of stimulation was unaffected by the epithelial Na⁺ channel (ENaC) inhibitor amiloride. The H₂O₂-induced Cl^- secretion was significantly inhibited by indomethacin as well as by mPGES-1 deficiency. Like H₂O₂, PGE₂ treatment induced a 2-fold increase in Isc that was reduced by the protein kinase A (PKA) inhibitors H-89 and KT5720. These data suggest that H₂O₂ stimulates CFTR Cl^- channel-mediated Cl^- secretion through COX- and mPGES-1-dependent release of PGE₂ and subsequent activation of PKA.