Hypoxia is widespread in solid tumors as a consequence of poorly structured tumor-derived neovasculature. Direct measurement of low oxygen levels in a range of adult tumor types has correlated tumor hypoxia with advanced stage, poor response to chemotherapy and radiotherapy, and poor prognosis. Little is known about the importance of hypoxia in pediatric tumors; therefore, we evaluated the effects of hypoxia on the response of the neuroblastoma cell lines SH-EP1 and SH-SY5Y to the clinically relevant drugs, vincristine, etoposide, and cisplatin. Short periods of hypoxia (1% O₂) of up to 16 hours had no effect on drug-induced apoptosis or clonogenic survival. Prolonged hypoxia of 1 to 7 days leads to reduction in vincristine- and etoposide-induced apoptosis in SH-SY5Y and SH-EP1 cells, and this was reflected in increased clonogenic survival under these conditions. Neither short-term nor prolonged hypoxia had any effect on the clonogenic response to cisplatin in SH-SY5Y cells. Hypoxia-inducible factor-1 (HIF-1) α was stabilized in these cell lines within 2 hours of hypoxia but was no longer detectable beyond 48 hours of hypoxia. Up-regulation of carbonic anhydrase IX showed HIF-1α to be transcriptionally active. Down-regulation of HIF-1α by short hairpin RNA interference and the small-molecule 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole reduced hypoxia-induced drug resistance. These results suggest that prolonged hypoxia leads to resistance to clinically relevant drugs in neuroblastoma and that therapies aimed at inhibiting HIF-1α function may be useful in overcoming drug resistance in this tumor. [Mol Cancer Ther 2006;5(9):2241–50]