Tumour necrosis factor-α (TNFα) and interleukin 1β (IL-1β) are implicated in inflammation-associated disturbances in vascular reactivity (1). The present study aims to determine the mechanism of impairment of pulmonary artery endothelial-dependent relaxation by TNFα and IL-1β. Male Wistar rats (290 g) were humanely killed by cervical dislocation. Pulmonary artery branches were dissected, cut into 2 mm rings and suspended in Krebs’ buffer for measurement of changes in tension. After precontraction with phenylephrine, acetylcholine (ACh) was added before and after 2h incubation with or without cytokines. In some experiments the selective iNOS inhibitor 1400W (1µM), the superoxide dismutase mimetic tempol (1mM), the NADPH oxidase inhibitor apocynin (300μM), the xanthine oxidase inhibitor allopurinol (1mM) or the eNOS cofactor tetrahydrobiopeptin BH4 (3μM) were added 30 min before the second addition of ACh. The changes in maximal relaxation (Emax) and pEC50 due to incubation were compared between treatments using ANOVA followed by Newman-Keuls’ post-hoc tests. Following treatment with TNFα (1ng.ml^-1, 2h), Emax values for ACh-induced relaxation of the pulmonary artery were reduced by 28.8±6.1 % compared with a reduction of 6.9±5.7 % in matched controls (p<0.05), and pEC50 values were increased by 1.03±0.17 compared with 0.35±0.07 log units in controls (p<0.001). These TNFα-induced changes were prevented by preincubation with either 1400W, tempol, BH4 or apocynin, resulting in Emax changes of 5.1 ± 3.7; 7.2 ± 5.0 and 1.1 ± 6.5 % in the case of tempol, BH4 or apocynin respectively (all p<0.05 compared with TNFα alone) and pEC50 shifts of 0.45 ± 0.06 (p<0.01), 0.50 ± 0.11 (p<0.05) and 0.51 ± 0.04 log units (p<0.01) respectively compared with TNFα alone. Preincubation with allopurinol had no significant effect on TNFα induced changes. Treatment of pulmonary artery rings with IL-1β (1ng.ml^-1) also led to a reduction in Emax (31.5±4.2 compared with 5.6 ± 1.8 % in controls, p<0.001) and an increase in the pEC50 shift (1.08±0.19 compared with 0.19 ± 0.05 log units in controls, p<0.001). These IL-1β-induced changes were prevented by preincubation with tempol or apocynin resulting in Emax changes of 10.5 ± 5.7 (p<0.05) and 2.8 ± 4.9% (p<0.01) respectively and a pEC50 shift of 0.29 ± 0.21 log units (p<0.01) in the case of apocynin compared with IL-1β alone. Preincubation with 1400W, BH4 or allopurinol had no significant effect on IL-1β induced changes. In conclusion, TNFα produces its effect through stimulation of NADPH oxidase and iNOS leading to eNOS uncoupling. IL-1β stimulates superoxide release via NADPH oxidase; however iNOS and eNOS uncoupling do not have a major role in the IL-1β induced impairment.