Native amiloride-sensitive Na+ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of δ-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na+ conductances in epithelial cells. For example, the δ-subunit may form multimeric channels with αβγ-ENaC. Reverse transcription-PCR revealed that δ-ENaC is co-expressed with αβγ-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that δ-ENaC is co-expressed with α-, β-, and γ-ENaC in H441 cells at the protein level. Measurement of current-voltage relationships revealed that the cation selectivity ratios for Na+/Li+/K +/Cs+/Ca2+/Mg2+, the apparent dissociation constant (Ki) for amiloride, and unitary conductances for δαβγ-ENaC differed from those of both αβγ- and δβlγ-ENaC (n = 6). The contribution of the δ subunit to PLi/PNa ratio and unitary Na+ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC50 for proton activation, mean open and closed times, and the self-inhibition time of δαβ γ-ENaC differed from those of αβγ- and δβγ-ENaC. Co-immunoprecipitation of δ-ENaC with α- and γ-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of δ-ENaC with other subunits could account for heterogeneity of native epithelial channels. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.