We investigated acute and chronic effects of hyperosmolality on mRNA and protein expressions of Na-K-ATPase α and β isoforms and Na-K-ATPase activity in the rat inner medullary collecting duct (IMCD). Incubation of IMCD in hypertonic medium for 30 min reduced the Na-K-ATPase activity by 50%. The Na-K-ATPase activity of dehydrated rats measured in isotonic medium was decreased, and incubation in hypertonic medium did not further decrease the activity. Incubation of IMCD in hypertonic medium for 6 h did not change α<sub>1</sub> mRNA. In contrast, dehydration decreased α<sub>1</sub> subunit mRNA and protein and β<sub>1</sub> protein expressions without changing β<sub>1</sub> mRNA. These data show (1) that acute hyperosmolality decreases Na-K-ATPase activity in IMCD without changing α<sub>1</sub> and β<sub>1</sub> mRNA and (2) that 2 days of dehydration decreased Na-K-ATPase activity by reducing α<sub>1</sub> and β<sub>1</sub> proteins. Thus, the mechanisms for the inhibition of the Na-K-ATPase activity in IMCD is defferent between acute and chronic exposure to hyperosmolality.