Hyperbaric oxygen (HBO) therapy is successfully used to treat chronic wounds by stimulating tissue repair, including angiogenesis. Angiogenesis usually involves the stimulation of VEGF production in blood vessels, possibly via NO signalling, but the role of HBO in this process is controversial. This study aimed to investigate the effect of HBO on VEGF and NO production by blood vessels in vitro. Male Sprague Dawley rats (350 – 400 g; n = 17) were killed by Sagatal (90 mg.kg^-1; I.P.). Sections of aorta were placed in culture dishes containing 6 ml of modified Krebs-Ringer solution (in mM: NaCl 118.6, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.1, Hepes 10, glucose 10, pH 7.4) for 30 min before 90 min treatments. The treatments were air at 1 ATA (control), 100% O₂ at 1 ATA or 100% O₂ at 2.2 ATA. After treatment the tissue remained in Krebs-Ringer for a further 4 hr in air. All experiments were conducted at 37 °C. Tissue and medium samples were collected pre-treatment (pre), immediately post treatment (post), and 4 hr after treatment (4h). We measured LDH activity, [lactate], [NO₂^–] and [H₂O₂] in the medium; [VEGF] in the medium and the tissue by ELISA (R&D Systems, MMV00). All data are means ± S.E.M (n = 7-8) and analyzed using ANOVA. LDH release and lactate content increased over time, and were lower in the HBO group during and following treatment when compared with either the control or oxygen only groups. At 4h, LDH in the HBO group (11.4 ± 2.7 U.g^-1 protein) was significantly lower (P < 0.05) than that in the control group (23.8 ± 10.5). Lactate in the HBO group at post and 4h (0.16 ± 0.02 and 0.44 ± 0.6 _μmol.mg^-1 protein) were significantly lower (P < 0.05) than the control (0.26 ± 0.03 and 0.72 ± 0.15). H₂O₂ remained at nanomolar level and showed a transient rise peaking at post, and then declining (not statistically significant between treatments). NO₂^– levels were stable throughout the experiment in all groups (range from 1.67 to 2.13 nmol.mg^-1 protein). [VEGF] in the medium remained at or below the detection limit (<3 pg.ml^-1). VEGF in crude homogenates of blood vessels was easily detected and progressively increased reaching peak values by 4h (34.2 ± 4.4, 38.0 ± 4.5, 33.5 ± 3.5 pg.mg^-1 protein in controls, oxygen only and HBO groups vs. 15.7 ± 5.5 pre level; P < 0.05), no treatment differences were shown. Overall we conclude that HBO does not damage the blood vessels as evidenced by low LDH leak and only nanomolar levels of H₂O₂ generation. No evidence for NO-induced VEGF release by a single HBO is shown from this study.