The epithelial sodium channel (ENaC) is a key mediator of sodium transport in epithelia; however, little is known about ENaC expression in mammary epithelia. Using real-time PCR, we demonstrated the expression of the ENaC subunit mRNAs in mouse and human mammary cell lines and in vivo mouse mammary tissue. We determined the effects of glucocorticoids, progesterone, and prolactin on ENaC expression in four mammary cell lines. Dexamethasone induced all detectable ENaC subunits in noncancerous cell lines, HC11 and MCF10A. Interestingly, in cancerous cell lines (T-47D and MCF-7), both beta- and gamma- but not alphaENaC mRNAs were induced by dexamethasone. Progesterone induced ENaC mRNA only in T-47D cells, and prolactin had no effects. gammaENaC was rapidly induced by steroids, whereas induction of alpha- and betaENaC was slower; moreover, the induction of the beta-subunit required de novo protein synthesis. Dexamethasone treatment did not affect ENaC mRNA stability. Western blot analysis revealed immunoreactive bands corresponding to different forms of alpha-, beta-, and gammaENaC; dexamethasone significantly increased the intensity of alphaENaC (85 kDa) and betaENaC (90 kDa). We also showed an in vivo reduction in alphaENaC levels in the mammary tissue of lactating mice as compared with controls, whereas beta- and gammaENaC mRNA levels were significantly increased. Furthermore, dexamethasone in vivo significantly increased alpha-, beta-, and gammaENaC mRNA expression. Our data indicate that both mouse and human mammary cells express all ENaC subunits, and they are regulated by steroid hormones in a temporal and cell-specific manner both in culture and in vivo.