Aim: The aim of this study was to determine the mechanisms of vasorelaxation in aortic rings in response to ACh and/or reduced pO2 and the role of oxidative stress and HIF-1α in acute and intermittent HBO2 protocols compared to untreated animals. Introduction: Hyperbaric oxygenation (HBO2) is used as adjuvant therapy in condition where tissue oxygen delivery is impaired. It is known that changes in pO2 may affect the synthesis of metabolites of arachidonic acid and thus vascular reactivity in health and disease, by involving CYP450 metabolites production. Generally, in healthy animals, vasorelaxationin response to hypoxia is mediated by prostacyclin (PGI2), while vasorelaxation in response to acetylcholine (ACh) is mainly mediated by NO pathway. Data on how is HIF-1α involved in relaxation mechanism are very scarce, inadequate and contradictory. Methods: Healthy Sprague-Dawley 9-12 weeks old rats were divided in four experimental groups (1. healthy control; 2. acute HBO2 group exposed to HBO2 (A-HBO2); 3. 24 hour after acute exposure (24H-HBO2); and 4. four subsequent days of HBO2 exposure (intermittent, 4D-HBO2). Reactivity to ACh and reduced pO2was tested in norepinephrine-precontracted aortic rings in the absence or in the presence of L-NAME, INDO, MS-PPOH, superoxide scavenger TEMPOL and LW6 HIF-1α inhibitor in tissue bath. The indicators of oxidative stress were measured by TBARS and FRAP methods and by direct measurement of endothelial superoxide production. Expression of antioxidative genes and enzyme-targeted genes was performed using RT PCR and proteins expression measurement was done by the Western blot. Results: In A-HBO2 group, HBO2 leads to impaired vasorelaxation response to both ACh and hypoxia, restored by TEMPOL in vitro to levels similar to control group vasorelaxation. ACh-induced vasorelaxation was mediated mainly by NO, with the contribution of CYP450 vasodilator metabolites in HBO2 groups respectively. Relaxation in response to reduced pO2 was partially blocked by both, INDO or MS-PPOH and not affected by L-NAME in all groups. HIF-inhibitor on functional level affected only relaxation in 24H-HBO2 group. TBARS and endothelial superoxide production were increased in A-HBO2 group compared to other tested groups. Conclusion: Acute exposure to HBO2 causes transient and reversible impaired relaxation of aortic rings, which is most likely caused by increased endothelial production of superoxide and overall increased oxidative stress. Intermitent exposure causes improved relaxation most likely by the activation of the CYP450 epoxygenase pathway of arachidonic acid metabolism and by increasing the generation and sensitivity to EETs.