Bovine pulmonary artery endothelial cells (BPAEC) are extremely sensitive to oxygen, mediated by superoxide production. Ionizing radiation is known to generate superoxide in oxygenated aqueous media; however, at systemic oxygen levels (3%), no oxygen enhancement is observed after irradiation. A number of markers (cell growth, alamarBlue, mitochondrial membrane polarization) for metabolic activity indicate that BPAEC maintained under 20% oxygen grow and metabolize more slowly than cells maintained under 3% oxygen. BPAEC cultured in 20% oxygen grow better when they are transiently transfected with either manganese superoxide dismutase (MnSOD) or copper zinc superoxide dismutase (CuZnSOD) and exhibit improved survival after irradiation (0.5–10 Gy). Furthermore, X irradiation of BPAEC grown in 20% oxygen results in very diffuse colony formation, which is completely ameliorated by either growth in 3% oxygen or overexpression of MnSOD. However, MnSOD overexpression in BPAEC grown in 3% oxygen provides no further radioprotection, as judged by clonogenic survival curves. Radiation does not increase apoptosis in BPAEC but inhibits cell growth and up-regulates p53 and p21 at either 3% or 20% oxygen.