Epithelial sodium channel (ENaC) blockers have been proposed as a therapy to restore mucus clearance (MC) in cystic fibrosis (CF) airways. The therapeutic effects of the first generation ENaC blocker, amiloride, in CF patients, however, were minimal. Because the failure of amiloride reflected both its low potency and short duration of action on airway surfaces, we investigated whether the increased potency of benzamil and phenamil would produce more favorable pharmacodynamic properties. In vitro potency, maximal efficacy, rate of recovery from maximal block of ENaC, and rate of drug absorption were compared for amiloride, benzamil, and phenamil in cultured human and ovine bronchial epithelial cells. In both human and ovine bronchial epithelia, the rank order of potency was benzamil > phenamil » amiloride, the maximal efficacy was benzamil = phenamil = amiloride, the recovery to baseline sodium transport was phenamil < benzamil « amiloride, and the rate of drug absorption was phenamil > benzamil » amiloride. Based on greater potency, benzamil was compared with amiloride in in vivo pharmacodynamic studies in sheep, including tracheal mucus velocity (TMV) and MC. Benzamil enhanced MC and TMV, but acute potency or duration of effect did not exceed that of amiloride. In conclusion, our data support the hypothesis that ENaC blocker aerosol therapy increases MC. However, rapid absorption of benzamil from the mucosal surface offset its greater potency, making it equieffective with amiloride in vivo. More potent, less absorbable, third generation ENaC blockers will be required for an effective aerosol CF pharmacotherapy.