Cellular iron is needed for cell survival and hydroxylation of hypoxia-inducible factor-1alpha (HIF-alpha) by prolyl hydroxylases (PHD). One mechanism of iron uptake is mediated by the cell surface transferrin receptor (TfR). Because iron is required for cell growth and suppression of HIF-alpha levels, we tested the effects of the two anti-TfR monoclonal antibodies (mAb) E2.3 and A27.15 on growth of breast cancer cells and induction of HIF-alpha and hypoxia-regulated genes. Treatment with both mAbs together synergistically inhibited cell proliferation in a dose-responsive manner by up to 80% following 8 days of exposure, up-regulated HIF-1alpha and HIF transcription targets, down-regulated TfR expression, and down-regulated cellular labile iron pool by 60%. Because combined treatment with anti-TfR mAbs resulted in the up-regulation of the hypoxia pathway, which may increase tumor angiogenesis, we analyzed the effects of ascorbate on cell viability and HIF-1alpha levels in cells treated with both anti-TfR mAbs together, as ascorbate has been shown to be required by PHD enzymes for full catalytic activity. Ascorbate at physiologic concentrations (25 micromol/L) suppressed HIF-1alpha protein levels and HIF transcriptional targets in anti-TfR mAb-treated cells but did not suppress the antiproliferative effect of the mAbs. These results indicate that the addition of ascorbate increased the activity of the PHD enzymes in down-regulating HIF but not the proliferation of iron-starved anti-TfR mAb-treated cells. The use of anti-TfR mAbs and ascorbate in inhibiting both cell proliferation and HIF-1alpha and angiogenesis under normoxic conditions may be of therapeutic use.