Introduction: Our previous study showed that acute exposure to hyperbaric oxygen (HBO2) increase oxidative stress parameter leading to impaired vasorelaxation to acetlycholine (ACh), while it was preserved in 4 days intermittent HBO2 protocols (1). Vasodilation in response to hypoxia is mainly mediated by cyclooxygenase (COX) activation and production of prostacyclin (PGI2) (2,3). However, cytochrome P450-epoxygenase metabolites may contribute to vasodilation in healthy vessels (4). It is known that changes in pO2 may affect the synthesis of metabolites of arachidonic acid (5). The aim of this study was to examine if acute single HBO2 exposure affects the reactivity of isolated rat aortic rings to hypoxia and to investigate if there are changes in vasorelaxation mechanisms due to HBO2. Methods: Healthy 9-12 weeks old Sprague-Dawley rats were divided in 3 groups. Control group (n=18, no treatment), A-HBO2 (n=16, acute HBO2 group exposed to HBO2 single 120′ session of 100% O2 at 2.0 bars with additionally 15′ for gradual compression and decompression) and A-HBO2+24H (n=19, rats tested 24 h after HBO2 exposure). Prior to decapitation and aorta preparation, rats were anesthetized with ketamin-chlorid (75 mg/kg) and midazolam (0.5 mg/kg). Response to reduced pO2 (bath gas mixture from 95% O2, 5% CO2 switched to bath gas mixture of 95% N2, 5% CO2) were tested in norepinephrine-precontracted aortic rings in the absence or in the presence of the NOS inhibitor L-NAME, COX-1,2 inhibitor indomethacin (INDO), MS-PPOH and superoxide scavenger TEMPOL in tissue bath. Thiobarbituric Acid Reactive Substances (TBARS) assay was used to determine oxidative stress in arterial blood samples. To test differences among groups One-way ANOVA or student t-test was used when appropriate (SigmaPlot v11.2, Systat Software, Chicago, USA), p<0.05 was considered significant. All experimental procedures conformed to the European Guidelines for the Care and Use of Laboratory Animals (directive 86/609) and were approved by the local and national Ethical Committee #2158/61-02-139/2-06. Results: TBARS was increased in A-HBO2 group compared to control and HBO2-24H group (p<0.05). Vasorelaxation in response to hypoxia was impaired in A-HBO2 group compared to other groups and was restored by TEMPOL in vitro to levels similar to control group vasorelaxation. Relaxation in response to hypoxia was not affected by L-NAME in any of tested groups, but was partially blocked by both, INDO or MS-PPOH in all groups. Conclusion: Acute HBO2 increases oxidative stress which may contribute to impaired vasorelaxation in A-HBO2 group. Both, COX and CYP450 pathways are activated and mediate hypoxia-induced vasorelaxation of aortic rings, irrespectively of treatment.