The effect of ventilation on movement of surfactant in airways was studied in rat lungs. Male albino rats were anesthetized and sacrificed by exsanguination. The trachea was cannulated and the diaphragm opened to allow the lungs to collapse. The lungs were left in chest and kept at minimal volume, or at constant inflation with air, or were ventilated with air, nitrogen or oxygen. At the end of 3 hr the extrapulmonary airways (EPA), i.e. trachea and bronchi, were separated from the lungs and flushed with 40 ml isotonic saline. The surface tension and lecithin content of the saline wash was then measured. The surface activity and lecithin content of the EPA wash did not change if the lungs were kept at minimal volume or at constant inflation, but increased following ventilation. This increase was directly related to the duration of ventilation and to the tidal volume and inversely related to the end-expiratory pressure. It was independent of O2 concentration of the gas used for ventilation. The results suggest that ventilation enhances the movement of surfactant in airways and that positive end-expiratory pressure prevents this effect of ventilation.