In addition to increased fluid filtration, impaired alveolar fluid reabsorption (AFR) may contribute to the formation of hydrostatic lung edema in left-sided heart disease. Recently, we identified pressure-induced endothelial NO formation as a potential mechanism inhibiting AFR. Here, we tested whether the absence of endothelial-derived NO preserves AFR. In isolated perfused lungs of male eNOS^-/-and wild-type mice, left atrial pressure (P_LA ) was elevated from 2 to 6 cmH₂O for 25 min. Resulting hydrostatic lung edema was determined as wet/dry ratio. To separate filtration from fluid absorption effects, both fluid fluxes were quantified at elevated P_LA of 15 cmH₂O in isolated perfused rat lungs by a double-indicator dilution technique. In wild-type control mice, P_LA elevation from 2 to 6 cmH₂O increased wet/dry ratio from 4.12 ± 0.16 to 10.11±0.40 (n=6 each, p<0.05). In eNOS^-/-mice, formation of hydrostatic lung edema was attenuated (wet/dry ratio 6.3 ± 0.23; n=6, p<0.05 vs. control). A similar reduction of wet/dry ratios to 6.51 ± 0.39 was achieved by perfusing wild-type mouse lungs with L-NAME, an NO synthesis inhibitor (n=6, p<0.05 vs control). In rat lungs, L-NAME increased AFR to 1.69 ± 0.76 ml/h as compared to -1.74 ± 1.06 ml/h in controls (n=7, p<0.05) while fluid filtration flux remained constant (control: 2.14 ± 0.62 ml/h vs. L-NAME: 2.72 ± 0.81 ml/h). Our data show that eNOS-derived NO inhibits AFR and thus, plays a pivotal role in hydrostatic lung edema formation.