Fluid clearance from the lung is critical for breathing to begin at birth and defects in this process can cause lasting impairment of lung function. This process is stimulated by a rise in circulating glucocorticoids before birth which drive expression and recruitment of epithelial Na⁺ channel (ENaC) subunits to the epithelial membrane. Previous work has shown that the glucocorticoid effect is, however, malleable, and can be augmented by other hormones such as insulin (1). Thus, kinases in the insulin signalling cascade must be involved in promoting glucocorticoid-dependent ENaC expression. The aim of this study was to determine if the mammalian target of rapamycin (mTOR), a kinase involved in insulin signalling, participated in the insulin-evoked amplification of glucocorticoid regulated Na⁺ transport. We focussed on the expression of αENaC as the major pore forming subunit of the ENaC channel. H441 cells maintained under serum free conditions were transfected with a luciferase reporter gene promoted by 2.3kb of the proximal αENaC promoter. A mutant form of the reporter in which the glucocorticoid response element had been mutated was used as a negative control. Cells were treated with either varying concentrations of dexamethasone (Dex; 0-200nM) or a single concentration of Dex (100nM) plus varying concentrations of insulin (0-1000nM) for 6 hours before harvesting. Luciferase assays revealed the activity of the αENaC promoter and western blotting was performed to determine total cellular αENaC abundance and mTOR activity (phosphorylation of p70S6K-T389). For statistical tests, data were analysed by one-way ANOVA with the level of significance determined by Tukey’s test. We found that Dex significantly increases the expression of αENaC, with the average fold change over control 4.94 ± 1.08 SEM (p value = 0.01 at 100nM Dex), and that insulin significantly augments this response (p value = 0.03 at 100nM insulin) with the average fold change over the Dex response 2.8 ± 0.65 SEM. Densometric analysis revealed a significant increase in the phosphorylation of p70S6K (p value = 0.03 at 100nM insulin). Total cellular abundance of αENaC was also increased by insulin (fold change over Dex response = 7.8 at 1nM of insulin). Rapamycin antagonised the insulin effect but failed to alter the expression of αENaC induced by Dex alone. Insulin significantly augments the Dex effect on H441 cells resulting in the increased activity of mTOR. This is paralleled by an increase in the total abundance of αENaC. We suggest that mTOR, a nutrient sensitive kinase, might play a role in the hormonal regulation of ENaC. This may have implications in fluid clearance at birth and also in any tissue expressing ENaC and in the treatments of any diseases resulting from defects of Na⁺ homeostasis.