Hypoxia and biological responses to hypoxia are commonly encountered in both normal and pathologic cellular processes. Here we report that extracellular superoxide dismutase (EC-SOD) plays a major role in regulating the magnitude of hypoxia-induced erythropoietin (Epo) gene expression, thus implicating superoxide as an intermediary signal transduction molecule critical to this process. We found that mice which have the EC-SOD gene inactivated show a marked more than 100-fold elevation in hypoxia-induced Epo gene expression, compared with wild-type controls, which was both dose and time dependent. These mice also showed a significant increase in serum Epo levels after 1 d hypoxia. Interestingly, despite elevated Epo levels, reciprocal changes in hematocrit and reticulocyte counts were not found, suggesting that this newly synthesized Epo lacks functional hematopoietic effects. When EC-SOD was overexpressed in Hep3B cells, we found a significant reduction in Epo gene induction by both CoCl2 (50 microM) and hypoxia (1% O2). Similar findings were noted with another hypoxia-inducible gene, carbonic anhydrase IX. We conclude that EC-SOD functions as a major repressor of hypoxia-induced Epo gene expression, which implicates superoxide as a signaling intermediate whose downstream effects, at least in part, may be mediated by HIF-1alpha.