We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human α-ENaC (αhENaC) led to a decrease and increase in the macroscopic conductance of αR586E,R587Eβγ and αR589E,R591Eβγ-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both αR586E,R587Eβγ and αR589E,R591EPβγ-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for αR586E,R587Eβγ-hENaC and increased for αR589E,R591Eβγ-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for αR586E,R587Eβγ-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.