The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's Syndrome) and impaired mucociliary clearance (in Cystic Fibrosis). The regulation of ENaC by molecular chaperones, such as the 70 kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggested that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes, we investigated the mechanism by which Hsc70 acts upon ENaC in epithelial cells. In MDCK cells stably expressing epitope-tagged αβ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 μg/ml Doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating both ENaC biogenesis and ENaC trafficking at the cell surface.