Limited information is available regarding domains within the epithelial Na؉ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278 -283) within the extracellular domain of ␣rENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant ␣rENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type ␣rENaC was inhibited by amiloride with a K i of 169 nM. Deletion of the entire WYRFHY tract (␣rENaC ⌬278 -283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (K i ؍ 26.5 M). Similar results were obtained when either ␣rENaC or ␣rENaC ⌬278 -283 were co-expressed with wt -and ␥rENaC (K i values of 155 nM and 22.8 M, respectively). Moreover, ␣rENaC H282D was insensitive to submicromolar concentrations of amiloride (K i ؍ 6.52 M), whereas ␣rENaC H282R was inhibited by amiloride with a K i of 29 nM. These mutations do not alter ENaC Na ؉ :K ؉ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of ␣rENaC and mutations preceding the second membrane-spanning domains of ␣-, -, and ␥rENaC alters amiloride's K i , suggest that selected regions of the extracellular loop of ␣rENaC may be in close proximity to residues within the channel pore.The diuretic amiloride is a prototypic inhibitor of epithelial Na ϩ channels (ENaCs) 1 (1), although amiloride and its various derivatives inhibit many Na ϩ -selective transport proteins. Several laboratories have recently identified domains within the epithelial Na ϩ channel and the Na ϩ /H ϩ exchanger that appear to participate in amiloride binding. Residues within the second membrane-spanning domain of ␣rENaC may interact with amiloride, as mutations of a serine residue at position 589 result in a large decrease of the apparent K i for amiloride and the amiloride analog benzamil, as well as alter cation selectivity (2). Selected mutations of residues within a hydrophobic region, termed H2 (3), immediately preceding the second membrane-spanning domains of the ␣-, -, and ␥-subunits of rENaC (i.e. Trp-␣582, Ser-␣583, Gly-525, Gly-␥537) and the ␣-subunit of bovine ENaC (Lys-504, Lys-515) affect the K i for amiloride, and several of these mutations affect single-channel conductance (4, 5). Snyder and co-workers have identified splice variants of ␣rENaC in which the C-terminal 199 or 216 amino acid residues, including the second membrane-spanning domain, are truncated (6). These splice variants are not functional when expressed in Xenopus oocytes, but retain amiloride and phenamil binding activity, s...