To assess the action of antidiuretic hormone (ADH) and the osmolality of bathing solution on amiloride-sensitive Na+ transport, we measured the amiloride-sensitive short-circuit current (Isc) and single-channel currents in renal epithelial A6 cells. The A6 cells were cultured on permeable supports for 9-13 d without aldosterone treatment. The basal amiloride-sensitive Isc and the density of the amiloride-sensitive Na+ channel at the apical membrane increased as the osmolality of the bathing solution decreased. ADH stimulated the amiloride-sensitive Isc. The stimulatory action of ADH was enhanced by low osmolality. The stimulatory action of hyposmolality on the amiloride-sensitive Isc was significantly diminished by pretreatment with brefeldin A (BFA, a blocker of protein translocation), while BFA had no significant effect on the ratio of ADH-stimulated amiloride-sensitive Isc to basal amiloride-sensitive Isc. These results suggest that hyposmolality stimulates the translocation of amiloride-sensitive Na+ channels to the apical membrane from the cytosolic store sites of the channel, and that ADH may activate amiloride-sensitive Na+ channels pre-existing in the apical membrane or translocate the channel via BFA-insensitive pathways in a manner dependent on the osmolality of the bathing solution in aldosterone-untreated A6 cells, differently from aldosterone-treated A6 cells in which ADH stimulates the translocation of amiloride-sensitive Na+ channels via BFA-sensitive pathways.