We have shown that cAMP-dependent protein kinase (PKA) signaling is essential for early morphologic changes that occur in epithelial cell migration and repair. We hypothesize that chronic ethanol exposure causes down-regulation of PKA signaling in airway epithelial cells and reduces the ability of airway epithelial cells to spread and migrate during wound repair. Exposure of primary cultures of bovine bronchial epithelial cells to ethanol at the start of closure of mechanical wounds did not impair initial wound closure, consistent with the positive effect of ethanol on PKA activity. When ethanol was present for longer times, however, there was inhibition of PKA activity and impaired wound closure. Pretreatment of cells with 100 mM ethanol for 18 hours also made the cells unresponsive to stimuli of PKA that accelerate wound closure. Cell monolayers exposed to ethanol also did not increase migration in response to 1 μM isoproterenol (14 ± 5% wound closure vs. 68 ± 3% wound closure for cells treated with isoproterenol). No cell toxicity was observed with 100 mM ethanol treatment. We next examined the effect of ethanol on cell morphology during assays of attachment and spreading on collagen-coated dishes. Both 2 and 24 hr exposure to ethanol reduced cell surface area of attaching cells (26 ± 6% and 24 ± 5% respectively) compared to control cells and the number of attached cells (30 ± 3% and 39 ± 1% attachment respectively, compared to 48 ± 1% attachment for control cells). Ethanol reduces bronchial epithelial cell spreading and migration in in vitro wound repair assays.