Isoprostanes are chemically stable lipid peroxidation products of arachidonic acid, the quantification of which provides a novel approach to the assessment of oxidative stress in vivo. The main objective of this study was to quantify the urinary levels of isoprostaglandin F_{2 α} type III (iPF_{2 α}-III), an F₂-isoprostane, in patients with pulmonary hypertension (PHT) in comparison with healthy controls. The secondary objective was to test whether baseline iPF_{2 α}-III levels correlate to the reversibility of pulmonary hypertension in response to inhaled NO challenge. Urinary iPF_{2 α}-III levels were measured by gas chromatography-mass spectrometry in 25 patients with PHT, 14 of whom were investigated for response to inhaled NO challenge. Urinary iPF_{2 α}-III levels in PHT patients (225 ± 27 pmol/mmol creatinine) were 2.3 times as high as in controls (97 ± 7 pmol/mmol creatinine, p < 0.001). The mean pulmonary arterial pressure variation and the pulmonary vascular resistance variation in response to inhaled NO were correlated to basal iPF_{2 α}-III levels. This study shows that oxidative stress is increased in patients with pulmonary hypertension. Furthermore, iPF_{2 α}-III levels inversely correlate to pulmonary vasoreactivity. These observations are consistent with the hypothesis that free radical generation is involved in PHT pathogenesis.