Reissner's membrane epithelium forms much of the barrier that produces and sustains the large ionic differences between cochlear endolymph and perilymph. We have reported that Reissner's membrane contributes to normal cochlear function by absorbing Na⁺ from endolymph via amiloride-sensitive channels in gerbil inner ear. In the present study, we sought in mouse Reissner's membrane 1) to identify candidate genes involved in the Na⁺ transport pathway, 2) to determine whether their level of expression was regulated by the synthetic glucocorticoid dexamethasone and 3) to obtain functional evidence for the physiologic importance of these genes. Transcripts were present for the -, - and -subunits of ENaC; the corticosteroid receptors GR (glucocorticoid receptor) and MR (mineralocorticoid receptor); the GR agonist-regulator 11-HSD1; Na⁺ transport control components SGK1, Nedd4-2, WNKs; K⁺ channels and Na⁺,K⁺-ATPase. Expression of the MR agonist-regulator 11-HSD2 was not detected. Dexamethasone upregulated transcripts for - and -subunits of ENaC (~6 and ~3 fold), KCNK1 (~3 fold), 11-HSD1 (~2 fold), SGK1 (~2 fold) and WNK4 (~3 fold). Transepithelial currents from the apical to basolateral side of Reissner's membrane were sensitive to amiloride (IC50: ~0.7 µM) and benzamil (IC50: ~0.1 µM), but not EIPA (IC50: ~34 µM); amiloride-blocked transepithelial current was not immediately changed by forskolin/IBMX. The currents were reduced by ouabain, lowered bath Na⁺ (150 to 120 mM) and K⁺ channel blockers (XE991, Ba²⁺ and acidification from pH 7.4 to 6.5). Dexamethasone-stimulated current and gene expression were reduced by mifepristone but not spironolactone. These molecular, pharmacological and functional observations are consistent with Na⁺ absorption by mouse Reissner's membrane that is mediated by apical ENaC and/or other amiloride-sensitive channels, basolateral Na⁺,K⁺-ATPase and K⁺-permeable channels and that is under the control of glucocorticoids. These results provide an understanding and molecular definition of an important transport function of Reissner's membrane epithelium in the homeostasis of cochlear endolymph.