Hypertonic saline (HS) inhalation therapy benefits Cystic Fibrosis (CF) patients (8; 11). Surprisingly, these benefits are long-lasting and are diminished by the epithelial Na⁺ channel blocker amiloride (8). Our aim was to explain these effects. Human bronchial epithelial (hBE) cells from CF lungs were grown in inserts and were used in three experimental approaches: i) Ussing chambers to measure amiloride-sensitive short circuit currents (I_Na); ii) Continuous perfusion Ussing chambers; and iii) The airway surface of the inserts was exposed to a small volume (30 µl) of isosmotic or HS solution as the inserts were kept in their incubation tray and were subsequently used to measure I_Na under isosmotic conditions (near "thin-film" experiments) (35). HS solutions (660 mOsm/Kg H₂O) were prepared by adding additional NaCl to the isosmotic buffer. The transepithelial short-circuit current (I_SC), conductance (G_T) and capacitance (C_T) were measured by transepithelial impedance analysis (6; 33). Exposure to apical HS inhibited I_Na, G_T and C_T. The I_Na inhibition required 60 minutes of re-exposure to the isosmotic solution to recover 75%. The time of exposure to HS required to inhibit I_Na was < 2.5 minutes. Under near "thin-film" conditions apical exposure to HS inhibited I_Na but as osmotically-driven water moved to the apical surface, the aqueous apical volume increased leading to an amiloride-insensitive decrease in its osmolality and to recovery of I_Na that lagged behind the osmotic recovery. Amiloride significantly accelerated the recovery of I_Na following exposure to HS. Conclusions: Exposure to HS inhibits hBE I_Na and Amiloride diminishes this effect.