In cystic fibrosis (CF), the absence of functional CFTR leads to dysregulated Na(+) absorption across airway epithelia. We established an in vitro model of dysregulated Na(+) absorption by treating polarized normal human bronchial epithelial cells (HBEs) with nystatin (Nys), a polyene antibiotic that enables monovalent cations to permeate biological membranes. Acute mucosal Nys produced a rapid increase in short circuit current (I(sc)) that reflected increased transepithelial Na(+) absorption and required Na(+)/K(+)ATPase activity. The acute increase in I(sc) was associated with increased mucosal liquid absorption. Prolonged mucosal Nys treatment resulted in sustained Na(+) hyperabsorption, associated with increased mucosal liquid absorption in comparison with naïve (nontreated, kept under air-liquid interface conditions) or vehicle-treated cultures. Nys treatment was not toxic. Increased lactate accumulation in Nys-treated culture media suggested a higher metabolic rate associated with the higher energy demand for Na(+) transport. After chronic Nys treatment, the increased I(sc) was rapidly lost when the cultures were mounted in Ussing chambers, indicating that Nys could be rapidly removed from the apical membrane. Importantly, chronic Nys treatment promoted sustained mucosal liquid depletion and caused mucus dehydration, compaction, and adhesion to the apical surface of Nys-treated cultures. We conclude that mucosal Nys treatment of HBEs provides a simple in vitro model to recapitulate the Na(+) and volume hyperabsorptive features of CF airway epithelia.