In renal distal nephron and airway epithelial cells, adrenocortical steroids increase epithelial Na⁺ channel (ENaC) activity and also markedly increase the expression of the alpha 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 5-8 fold, endogenous -ENaC mRNA and protein about 10-fold, and -ENaC protein and mRNA 1.2-2 fold. -ENaC expression was unchanged. To determine if 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 and/or subunit had no effect on ENaC activity in collecting duct cells or lung epithelial cells. In contrast, a small increase in -ENaC expression increased ENaC activity about 3-fold. This increase in activity was additive to the effect of steroids. Thus, even though corticosteroids strongly increase -ENaC expression and moderately increase -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 -ENaC is necessary for the full steroid effect on ENaC. Increased expression of -ENaC and corticosteroid treatment enhance ENaC activity by parallel, non-interacting 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 -ENaC expression.