Oxidative stress may be responsible for the reduction in systemic nitric oxide (NO) bioavailability and impaired neurovascular reactivity recently observed during acute exposure to inspiratory hypoxia (Bailey et al., 2009). However, the brain’s contribution to these systemic changes in redox-homeostasis remains unknown. Therefore, the present study determined the trans-cerebral exchange kinetics of oxidative-nitrative stress biomarkers in response to hypoxia. We hypothesised that hypoxia would increase the net cerebral output of free radicals that would inactivate NO as indicated by a decrease in the net uptake or consumption of nitrite (NO₂‘) and increased output of 3-nitrotyrosine (3-NT). Ten healthy males aged 27 (mean) ± 4 (SD) years were examined in normoxia and following 9h passive exposure to hypoxia (12.9%O₂). Global cerebral blood flow (CBF) was measured using the Kety-Schmidt technique with paired samples obtained from the radial artery and internal jugular vein. Global cerebral plasma flow (CPF) was determined as CBF x (1-haematocrit). The serum concentration of spin-trapped α-phenyl-tert-butylnitrone (PBN)-adducts was assessed via X-band electron paramagnetic resonance spectroscopy. Plasma NO₂‘ was determined by ozone-based chemiluminescence using modified tri-iodide reagent and 3-NT via ELISA. Trans-cerebral net exchange was calculated as the arterio-jugular venous concentration difference x CPF. Data were analysed with a two-way repeated measures ANOVA and post-hoc Bonferroni-corrected paired samples t-tests. Despite a marked reduction in PaO₂ (107 ± 6 to 46 ± 3 mmHg, P < 0.05), the cerebral metabolic rate for O₂ was preserved (2.43 ± 0.54 in normoxia vs. 2.49 ± 0.34 in hypoxia, P > 0.05) due to an increase in global CBF [85 ± 15 in normoxia vs. (PaCO₂-corrected) 123 ± 24 mL/100g/min, P < 0.05). Hypoxia increased the net cerebral output of PBN-adducts identified as lipid-derived alkoxyl radicals (-73 ± 192 vs. -360 ± 253 AU/g/min, P < 0.05). This was associated with an attenuation in the net uptake of NO₂‘ (126.4 ± 93.9 vs. 16.0 ± 46.7 nmol/g/min, P < 0.05) and increased output of 3-NT (-3.1 ± 9.0 vs. -10.7 ± 18.2 nmol/g/min, P < 0.05). These findings provide the first direct evidence for increased oxidative-nitrative stress in the hypoxic human brain. The regional loss of NO₂‘ likely reflects the combined effects of NO “consumption” to support the observed increase in CBF to preserve cerebral O₂ delivery and NO “loss” subsequent to oxidative inactivation by superoxide.