Pulmonary vasoconstriction in response to alveolar hypoxia (HPV) is frequently impaired in patients with sepsis or the acute respiratory distress syndrome (ARDS) or in animal models of endotoxemia. It has been suggested that pulmonary vasodilation due to overproduction of nitric oxide (NO) by NO synthase 2 (NOS2) may be responsible for this impaired HPV following administration of endotoxin (LPS). We investigated the effects of acute non-specific (L-NAME) and NOS2-specific (L-NIL) NOS inhibition and of congenital deficiency of NOS2 on impaired HPV during endotoxemia. The pulmonary vasoconstrictor response and pulmonary vascular pressure-flow (P/Q) relationship during normoxia and hypoxia were studied in isolated, perfused, and ventilated lungs from LPS-pretreated and untreated wild-type and NOS2-deficient mice with and without L-NAME or L-NIL added to the perfusate. Compared with lungs from untreated mice, the lungs from LPS-challenged wild-type mice constricted less in response to hypoxia (69±17% versus 3±7%; respectively, P<0.001). Perfusion with L-NAME or L-NIL restored this blunted HPV response only in part. In contrast, LPS administration did not impair the vasoconstrictor response to hypoxia in NOS2-deficient mice. Analysis of the pulmonary vascular P/Q relationship suggested that the HPV response may consist of different components that are NOS isoform-specific modulated in untreated and LPS-treated mice. These results demonstrate in a murine model of endotoxemia, that NOS2-derived NO production is critical for LPS-mediated development of impaired HPV. Furthermore, we provide evidence that impaired HPV during endotoxemia is at least in part mediated by mechanisms other than simply pulmonary vasodilation by NOS2-derived NO overproduction.