Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays an important role in O(2) homeostasis. Numerous observations suggest that changes in reactive oxygen species affect HIF-1 alpha stabilization and HIF-1 alpha transcriptional activation in many cell types. The antioxidant enzyme manganese superoxide dismutase (MnSOD) modulates the cellular redox environment by converting superoxide (O(2)(*-)) to hydrogen peroxide and dioxygen. Previous results from our group have shown that overexpression of MnSOD in MCF-7 cells alters stabilization of HIF-1 alpha under hypoxic conditions; however, the underlying mechanism(s) is not known. Here, we tested the hypothesis that MnSOD regulates the expression of HIF-1 alpha by modulating the steady-state level of O(2)(*-). We found that decreasing MnSOD with small interfering RNA in MCF-7 cells resulted in (a) an associated increase in the hypoxic accumulation of HIF-1 alpha immunoreactive protein, (b) a significant increase in the levels of O(2)(*-) (P < 0.01), but (c) no significant change in the steady-state level of H(2)O(2). Removal of O(2)(*-) using spin traps (alpha-4-pyridyl-1-oxide-N-tert-butylnitrone and 5,5-dimethyl-1-pyrroline N-oxide) or the O(2)(*-) scavenger Tempol or an SOD mimic (AEOL10113) resulted in a decrease in HIF-1 alpha protein, consistent with the hypothesis that O(2)(*-) is an important molecular effector responsible for hypoxic stabilization of HIF-1 alpha. The evidence from both genetic and pharmaceutical manipulation is consistent with our hypothesis that O(2)(*-) can contribute to the stabilization of HIF-1 alpha.