Our previous studies using oxygen microelectrodes showed that the thymus is grossly hypoxic under normal physiological conditions. We now have investigated how oxygen tension affects the thymus at the cellular and molecular level. Adducts of the hypoxia marker drug pimonidazole accumulated in foci within the cortex and medulla and at the corticomedullary junction, consistent with the presence of widespread cellular hypoxia in the normal thymus. Hypoxia-associated pimonidazole accumulation was decreased but not abrogated by oxygen administration. Genes previously reported to be induced by hypoxia were expressed at baseline levels in the normal thymus, indicating that physiological adaptation to hypoxia occurred. Despite changes in thymus size and cellularity, thymic PO₂ did not change with age. Combined assays for hypoxia and cell death showed that hypoxia achieved using either hypoxic gas mixtures or high-density culture in normoxia decreased spontaneous thymocyte apoptosis in vitro. Taken together, these data suggest that regulatory mechanisms exist to maintain thymic cellular hypoxia in vivo and that oxygen tension may regulate thymocyte survival both in vitro and in vivo.