High tidal volume (HV_T) ventilation causes pulmonary endothelial barrier dysfunction. HV_T ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV_T lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V_T. Ventilation with 20 ml/kg V_T for 80 min increased the filtration coefficient (Kf), an index of vascular permeability. The increased cGMP and Kf caused by HV_T were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS (eNOS) knock-out mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 µM ODQ) also blocked cGMP generation and inhibited the increase in Kf, suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans Blue dye albumin extravasation and wet/dry ratio in an anesthetized mouse model of HV_T injury. Additional activation of sGC (1.5 µM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and Kf in lungs ventilated with 15 ml/kg V_T. HV_T endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 µM IBMX) as well as an inhibitor (10 µM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V_T-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV_T-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.