Purpose: Pulmonary hypertension plays a significant role in the pathophysiology of congenital diaphragmatic hernia (CDH). We hypothesized that nitric oxide-cGMP mechanisms of pulmonary vasodilation may be altered in CDH. We examined the vasoactive properties of pulmonary vessels in fetal lambs with surgically created CDH. Methods: Left sided diaphragmatic hernias were created in 8 fetal lambs at 78 days gestation (term 140-145 days), followed by operative delivery at 139 days. Fourth generation pulmonary arteries (PA) and veins (PV) were isolated from both left (ipsilateral) and right (contralateral) lungs, and studied using standard tissue bath techniques. Basal activity of NO synthase (NOS) in endothelium intact vessels was assessed by measuring the contractile force to norepinephrine in the presence and absence of the competitive NOS inhibitor LNA. Relaxations to A23187 (receptor independent agonist for NOS), SNAP (NO donor), and zaprinast(cGMP phosphodiesterase inhibitor) were then examined in preconstricted vessels. Results: Both left and right PA contractile responses to NE were enhanced compared to controls. Relaxations to A23187, SNAP, and zaprinast in PA isolated from CDH lambs were similar to control lambs. In contrast, relaxations to A23187 and zaprinast were blunted in both left and right PV from CDH lambs compared to controls. Relaxations were significantly more blunted in left compared to right PV. Further, left PV from CDH lambs did not display an enhanced contractile response to NE following LNA pretreatment, while right PV did. Relaxations to SNAP were equivalent to controls in both right and left PV. Conclusion: We conclude that the NO-cGMP pathway is abnormal in the near term lamb with CDH. These abnormalities are most apparent in PV, and may reflect abnormal vascular NOS activity and/or content between left and right lungs. PA have NOS activity similar to controls, but appear to be hypersensitive to exogenous vasoconstrictors due to other mechanisms.