The primary objective of these studies was to test the contribution of ventilation and blood flow to the removal of excess liquid from the air spaces and interstitium of the lung. First, after eliminating ventilation by clamping the left main bronchus in anesthetized sheep, alveolar and lung liquid clearance was not altered over 4 h compared with control sheep that were ventilated normally. Thus, removal of excess liquid across the alveolar epithelium was independent of the change in the transalveolar hydrostatic pressure gradient produced by ventilation. Second, to determine the effect of removing all blood flow to the lung, we developed a new in situ sheep lung model in which lung lymph flow was measured over 4 h with or without ventilation after the sheep had been exsanguinated. Alveolar liquid clearance, as measured by the percent increase in alveolar protein concentration over 4 h, was similar between sheep without blood flow (31 +/- 18%) compared with sheep with normal blood flow to the lungs (31 +/- 17%). Lung lymph flow contributed to only 10-15% of the clearance of the excess alveolar liquid that was transported to the interstitium, indicating that nonlymphatic pathways accounted for most of the excess lung liquid clearance in the absence of microvascular filtration. Third, because ouabain completely inhibited alveolar liquid clearance in this in situ sheep lung model, these data provide evidence that alveolar liquid clearance depends on an intact Na(+)-K(+)-ATPase-dependent pump mechanisms. Finally, this in situ model represents a unique experimental preparation that can be used to study the alveolar epithelial barrier without blood flow or ventilation for a short time (4 h) interval.