Ischemia-reperfusion (IR) causes human lung injury in association with the release of atrial and brain natriuretic peptides (ANP and BNP) but the role of ANP/BNP in IR lung injury is unknown. ANP and BNP bind to natriuretic peptide receptor A (NPR-A) generating cGMP, and to NPR-C, a clearance receptor that can decrease intracellular cAMP. To determine the role of NPR-A signaling in IR lung injury, we administered the NPR-A blocker anantin in an in-vivo SWR mouse preparation of unilateral lung IR. With uninterrupted ventilation, the left pulmonary artery was occluded for 30 min, and then reperfused for 60 or 150 min. Anantin administration decreased IR-induced Evans blue dye (EBD) extravasation and wet weight in the reperfused left lung suggesting an injurious role for NPR-A signaling in lung IR. In isolated mouse lungs, exogenous ANP (2.5 nM) added to the perfusate significantly increased the filtration coefficient (Kf) by 7-fold only if lungs were subjected to IR. This effect of ANP was also blocked by anantin. Unilateral in vivo IR increased endogenous plasma ANP, lung cGMP concentration and lung protein kinase G (PKG_I) activation. Anantin enhanced plasma ANP concentrations, attenuated the increase in cGMP and PKG_I activation, but had no effect on lung cAMP. These data suggest that lung IR triggered ANP release and altered endothelial signaling so that NPR-A activation caused increased pulmonary endothelial permeability.