T he mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia and aquaporin-4 (AQP4) in airway epithelia. To test whether these water channels facilitate fluid movement between airspace, interstitial, and capillary compartments, we measured passive and active fluid transport in AQP1 and AQP4 knockout mice. Airspace–capillary osmotic water permeability (P_f) was measured in isolated perfused lungs by a pleural surface fluorescence method. P_f was remarkably reduced in AQP1 (–/–) mice (measured in cm/s × 0.001, SE, n = 5–10: 17 ± 2 [+/+]; 6.6 ± 0.6 AQP1 [+/–]; 1.7 ± 0.3 AQP1 [–/–]; 12 ± 1 AQP4 [–/–]). Microvascular endothelial water permeability, measured by a related pleural surface fluorescence method in which the airspace was filled with inert perfluorocarbon, was reduced more than 10-fold in AQP1 (–/–) vs. (+/+) mice. Hydrostatically induced lung interstitial and alveolar edema was measured by a gravimetric method and by direct measurement of extravascular lung water. Both approaches indicated a more than twofold reduction in lung water accumulation in AQP1 (–/–) vs. (+/+) mice in response to a 5- to 10-cm H₂O increase in pulmonary artery pressure for five minutes. Active, near-isosmolar alveolar fluid absorption (J_v) was measured in in situ perfused lungs using ¹²⁵I-albumin as an airspace fluid volume marker. J_v (measured in percent fluid uptake at 30 min, n = 5) in (+/+) mice was 6.0 ± 0.6 (37°C), increased to 16 ± 1 by β-agonists, and inhibited to less than 2.0 by amiloride, ouabain, or cooling to 23°C. J_v (with isoproterenol) was not affected by aquaporin deletion (18.9 ± 2.2 [+/+]; 16.4 ± 1.5 AQP1 [–/–]; 16.3 ± 1.7 AQP4 [–/–]). These results indicate that osmotically driven water transport across microvessels in adult lung occurs by a transcellular route through AQP1 water channels and that the microvascular endothelium is a significant barrier for airspace–capillary osmotic water transport. AQP1 facilitates hydrostatically driven lung edema but is not required for active near-isosmolar absorption of alveolar fluid.