Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange. Chronic alveolar hypoxia results in vascular remodeling and pulmonary hypertension. Previous studies have reported conflicting results as to the effect of chronic alveolar hypoxia on pulmonary vasoreactivity and the contribution of NO, which may be related to species and strain differences as well as to the duration of chronic hypoxia. Therefore, we investigated the impact of chronic hypoxia on HPV features in rabbits, with particular focus on lung nitric oxide (NO) synthesis. After exposure of the animals to normobaric hypoxia (10% O₂) for 1 day up to 10 weeks, vascular reactivity was investigated in ex-vivo perfused normoxic ventilated lungs. Chronic hypoxia induced right heart hypertrophy and increased normoxic vascular tone within weeks. The vasoconstrictor response to an acute hypoxic challenge was strongly downregulated within 5 days, whereas the vasoconstrictor response to the thromboxane mimetic U46619 was maintained during the entire period of chronic hypoxia. The rapid down-regulation of HPV was apparently not linked to changes in the lung vascular NO system, detectable in the exhaled gas, because the amount of exhaled NO, the immediate drop of NO exhalation upon onset of the hypoxic maneuver, and the strong amplification of the hypoxic vasoconstrictor response upon blockade of NO synthesis by L-NMMA were fully maintained in the lungs originating from the chronic hypoxic rabbits. Treatment of the animals with long-term inhaled NO during the period of chronic hypoxia reduced right heart hypertrophy and partially maintained the reactivity to acute hypoxia challenge, without any impact on the endogenous NO system being noted. We conclude that chronic hypoxia causes rapid downregulation of the acute HPV response as a specific event, preceding the development of major pulmonary hypertension and being independent of the lung vascular NO system. Long-term NO inhalation partially maintains the strength of the hypoxic vasoconstrictor response.