Glucocorticoids and mineralocorticoids modulate Na⁺ transport via epithelial Na⁺ channels (ENaC). The rat submandibular epithelial cell line, SMG-C6, expresses α-ENaC mRNA and protein and exhibits amiloride-sensitive Na⁺ transport when grown in low-serum (2.5%) defined medium, therefore, we examined the effects of altering the composition of the SMG-C6 cell growth medium on ENaC expression and function. No differences in basal or amiloride-sensitive short-circuit current (I_sc) were measured across SMG-C6 monolayers grown in the absence of thyroid hormone, insulin, transferrin, or EGF. In the absence of hydrocortisone, basal and amiloride-sensitive I_sc significantly decreased. Similarly, monolayers grown in 10% serum-supplemented medium had lower basal I_sc and no response to amiloride. Adding hydrocortisone (1.1 μM) to either the low or 10% serum medium increased basal and amiloride-sensitive I_sc, which was blocked by RU486, the glucocorticoid and progesterone receptor antagonist. Aldosterone also induced an increase in α-ENaC expression and Na⁺ transport, which was also blocked by RU486 but not by the mineralocorticoid receptor antagonist spironolactone. Thus, in the SMG-C6 cell line, hydrocortisone and aldosterone increased ENaC expression and basal epithelial Na⁺ transport. The absence of endogenous ENaC expression in culture conditions devoid of steroids makes the properties of this cell line an excellent model for investigating pathways regulating ENaC expression and Na⁺ transport.