Cells exposed to oxygen deprivation in vitro have been shown to reduce proliferation and/or engage in programmed cell death. There is considerable controversy in the literature as to the role of hypoxia-inducible factor-1 (HIF-1) and HIF-1 target genes in initiating these responses. We therefore examined the oxygen dependence and the role of the hypoxia-responsive transcription factor HIF-1 in making the cellular death decision. Oxygen concentrations as low as 0.5% did not alter the growth of HIF-1–proficient or HIF-1–deficient murine fibroblasts, or human tumor cells, despite the appropriate induction of HIF-1 target genes. Severe hypoxia (<0.01% oxygen) did induced apoptosis, resulting in decreased colony formation, chromatin condensation, DNA fragmentation, and caspase activation but also independent of HIF1α status. Transcriptional induction of HIF-1–dependent genes putatively involved in cell death like BNip3 and BNip3L was therefore disassociated from hypoxia-dependent toxicity. Likewise, forced overexpression of a nondegradable form of HIF-1α in several human tumor cell lines was not sufficient to induce apoptosis under normoxic conditions. Taken together, these findings indicate that additional molecular events are triggered by anoxia in a HIF-1–independent manner, and these changes are necessary for cell death observed in low-oxygen environments.