Prostaglandin E₁ (PGE₁) is often added to the donor pulmonary flush solution to enhance clinical lung preservation for transplantation. Although PGE₁ is thought to act as a pulmonary vasodilator during the harvest period, the precise mechanism(s) of action whereby PGE₁ enhances lung preservation is unknown. Because cAMP levels decline in endothelial and vascular smooth muscle cells exposed to hypoxia, we hypothesized that a PGE₁-mediated increase in cAMP levels within the preserved lungs might improve pulmonary vascular homeostasis following lung transplantation. Rat lungs demonstrated a time-dependent decline in cAMP levels during hypothermic storage, with cAMP levels significantly increased by PGE₁ supplementation (≈2-fold by 6 hours, P<.0005). To test whether augmenting cAMP levels may enhance lung preservation, experiments were performed using an orthotopic rat left lung transplant model. Compared with controls, supplementing the preservation solution with the membrane-permeable cAMP analogue dibutyryl-cAMP resulted in dose-dependent preservation enhancement, marked by reduced pulmonary vascular resistance (6.0-fold, P<.01), improved arterial oxygenation (3.0-fold, P<.01), reduced graft neutrophil infiltration (1.5-fold, P<.05), and improved recipient survival (7.0-fold, P<.005). Similar preservation enhancement was observed with another cAMP analogue (8-bromo-cAMP) or the phosphodiesterase inhibitor indolidan. Stimulating the cAMP second messenger system by PGE₁ supplementation resulted in marked hemodynamic benefits and improved recipient survival, in parallel with reduced graft neutrophil infiltration, vascular permeability, and platelet deposition. These beneficial effects of PGE₁ were abrogated by simultaneous administration of the cAMP-dependent protein kinase antagonist Rp-cAMPS. Although an arterial vasodilator (minoxidil) resulted in significant pulmonary vasodilation during harvest, it lacked other nonvasodilating effects of PGE₁ and resulted in poor preservation. These data show that harvest vasodilation by itself is insufficient to enhance lung preservation and that PGE₁ enhances lung preservation by stimulating the cAMP-dependent protein kinase and promoting nonvasodilatory mechanisms of pulmonary protection.