Aldosterone and osmotic stress are well known to regulate the epithelial Na+ channel (ENaC)-mediated Na+ transport in renal epithelial cells. However, we have no information on how aldosterone and osmotic stress interact on stimulation of ENaC-mediated Na+ transport in renal epithelium. In the present report, we studied how application of aldosterone (1 mu M for 1 day) modifies the action of hypotonic stress on the ENaC-mediated Na+ transport in renal A6 epithelial cells by measuring the benzamil (a specific inhibitor for ENaC)-sensitive short-circuit current. The present study suggests that: (1) most ENaCs in cells without aldosterone treatment are translocated to Golgi apparatus, (2) major parts of aldosterone-generated ENaCs are located at the endoplasmic reticulum, (3) aldosterone diminishes the endocytosis rate of ENaCs from the apical membrane without any significant changes in the insertion rate of ENaCs into the apical membrane, and (4) application of sucrose after hypotonic stress stimulates the endocytosis of ENaCs, and elongates the functional life time of ENaCs by enhancing recycle of ENaCs into the endoplasmic reticulum in a retrograde manner. (C) 2007 Elsevier Inc. All rights reserved.