We have generated two site-directed mutants. K504E and K515E, in the α subunit of an amiloride-sensitive bovine epithelial Na+ channel, αbENaC. The region in which these mutations lie is in the large extracellular loup, immediately prior to the second membrane spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E αbENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channel exhibits either reduced or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ permeability, (from approximately 10:1 for wild-type αbENaC to 2:1 and 3.1 for K504E and K515E αbENaC, respectively), and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (K1) for amiloride, (K) values were 0.11±0.03 μM (n = 12). 0.54±0.10 μM (n = 6), and 0.95±0.18μM (n = 7), for wild-type, K515E, and K504E αbENaC, respectively). Both K504E and K515E αbENaC mutants were significantly more permeable to K+, as compared to wild-type αbENaC. Single channel conductance was not affected by either mutation. These observations identify a lysine-rich region between amino acid residues 495-516 of αbENaC as important to the regulation of fundamental channel properties.