Pulmonary production of NO increases in adult animals exposed to endotoxin. Neonatal endotoxemia is usually accompanied by pulmonary hypertension. The existence of pulmonary hypertension in the presence of increased NO production is inconsistent with NO's role as a vasodilator. We examined pulmonary NO production in neonatal endotoxemia by measuring exhaled NO and by comparing plasma NO metabolite concentration in paired pulmonary artery and pulmonary vein samples. Ten to fourteen day old piglets were anesthetized, tracheostomized and instrumented with pulmonary artery and systemic arterial catheters. Each piglet received 20 mcg/kg E. Coli lipopolysaccharide infused over 30 minutes. PVR, SVR, exhaled NO and paired PA and PV plasma NO metabolites were measured at 60 minute intervals. One hundred twenty minutes after endotoxin infusion was completed, the piglets were randomized to receive either a nitric oxide synthase inhibitor (L-NAME or aminoguanidine), the substrate for NO production (L-Arginine) or saline control. PVR increased significantly from baseline and was sustained throughout the 240 minute experimental period with no difference among groups after randomization. There was no change in peak exhaled NO over time in the control group or in those receiving L-NAME or aminoguanidine. Exhaled NO increased significantly in the two hours following administration of L-Arginine (4.41 ± 2.6 to 8.1± 3.8 ppb, p < 0.01). There was no difference in plasma NO metabolites over time within groups or between groups. We conclude that neonatal endotoxemia does not acutely increase pulmonary production of NO. We speculate that an inability to increase NO production may play a role in pulmonary hypertension associated with neonatal sepsis.