The amiloride-sensitive epithelial Na⁺ channel (ENaC), which is made of three different but homologous subunits, controls the rate of transepithelial Na⁺ absorption in a variety of epithelia. The present study investigated the functional role of its subunits in regulating ENaC activity, measured as amiloride sensitive short-circuit current (I_SC), in the mouse endometrial epithelium under different culture conditions. The treatment of the cultured epithelia with aldosterone (1 μM) or culturing cells on filters coated with concentrated Matrigel resulted in an increase in the amiloride-sensitive I_SC. Semiquantitative RT-PCR demonstrated that the expression of α and β subunits was not significantly altered by these treatments, but an increase in the γ subunit expression was observed. An 11-fold increase, induced by aldosterone, in the expression of the γ subunit, but not in the α and β subunits, was confirmed by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The treatment of endometrial cells with antisense against the γENaC subunit abolished the aldosterone-enhanced amiloride-sensitive I_SC. The results indicated an important role of γENaC subunit in determining ENaC activity, and a possible role of the γENaC subunit in interacting with CFTR was also discussed.