In renal distal nephron and airway epithelial cells, adrenocortical steroids increase epithelial Na⁺ channel (ENaC) activity and also markedly increase the expression of the α-subunit. The present experiments were designed to reconstitute this steroid effect in ENaC-expressing cells by overexpressing the subunits whose expression is enhanced by corticosteroids. In renal collecting duct monolayers, corticosteroids increased ENaC activity 5- to 8-fold, endogenous α-ENaC mRNA and protein ∼10-fold, and β-ENaC protein and mRNA 1.2- to 2-fold. γ-ENaC expression was unchanged. To determine whether this increase in expression was sufficient to increase ENaC activity, we used a regulated adenovirus system to increase expression of each subunit alone and in combination. Unexpectedly, increased expression of the α- and/or β-subunit had no effect on ENaC activity in collecting duct cells or lung epithelial cells. In contrast, a small increase in γ-ENaC expression increased ENaC activity about threefold. This increase in activity was additive to the effect of steroids. Thus, even though corticosteroids strongly increase α-ENaC expression and moderately increase β-ENaC expression, these effects are not, by themselves, sufficient to increase ENaC activity. Knockdown experiments are consistent with the idea that the increased expression of α-ENaC is necessary for the full steroid effect on ENaC. Increased expression of γ-ENaC and corticosteroid treatment enhances ENaC activity by parallel, noninteracting pathways. These results underscore the importance of other actions of steroid hormones for long-term enhancement of ENaC activity and raise new possibilities for regulation of ENaC activity by γ-ENaC expression.