Epithelial Na+ channels (ENaC) regulate salt and water re-absorption across the apical membrane of absorptive epithelia such as the kidney, colon, and lung. Structure-function studies have suggested that the second transmembrane domain (M2) and the adjacent pre- and post-M2 regions are involved in channel pore formation, cation selectivity, and amiloride sensitivity. Because Na+ selectivity, unitary Na+ conductance (γNa), and amiloride sensitivity of δ-ENaC are strikingly different from those of α-ENaC, the hypothesis that the pre-H2 domain may contribute to these characterizations has been examined by swapping the pre-H2, H2, and both (pre-H2+H2) domains of α- and α-ENaCs. Whole-cell and single channel results showed that the permeation ratio of Li+ and Na+ (PLi/PNa) for the swap α chimeras coexpressed with βγ-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of PLi/P Na for the swap δ constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the γNa of α-ENaC but decreased the γNa of δ-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (Kiamil) was observed in the α chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of Kiamil was obtained in the chimeric δ constructs with substitution of the H2 and pre-HE2+H2 domains. Our results demonstrate that the pre-H2 domain, combined with the H2 domain, contributes to the PLi+PNa ratio, single channel Na+ conductance, and amiloride sensitivity of α- and δ-ENaCs.