We investigated the mechanisms by which chlorine (Cl2) and its reactive byproducts inhibit Na+-dependent alveolar fluid clearance (AFC) in vivo and the activity of amiloride-sensitive epithelial Na+ channels (ENaC) by measuring AFC in mice exposed to Cl2 (0-500 ppm for 30 min) and Na+ and amiloride-sensitive currents (INa and Iamil, respectively) across Xenopus oocytes expressing human α-, β-, and γ-ENaC incubated with HOCl (1-2000 μM). Both Cl2 and HOCl-derived products decreased AFC in mice and whole cell and single channel INa in a dose-dependent manner; these effects were counteracted by serine proteases. Mass spectrometry analysis of the oocyte recording medium identified organic chloramines formed by the interaction of HOCl with HEPES (used as an extracellular buffer). In addition, chloramines formed by the interaction of HOCl with taurine or glycine decreased I Na in a similar fashion. Preincubation of oocytes with serine proteases prevented the decrease of INa by HOCl, whereas perfusion of oocytes with a synthetic 51-mer peptide corresponding to the putative furin and plasmin cleaving segment in the γ-ENaC subunit restored the ability of HOCl to inhibit INa. Finally, INa of oocytes expressing wild type α- and γ-ENaC and a mutant form of βENaC (S520K), known to result in ENaC channels locked in the open position, were not altered by HOCl. We concluded that HOCl and its reactive intermediates (such as organic chloramines) inhibit ENaC by affecting channel gating, which could be relieved by proteases cleavage. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.