Introduction: Hypoxia is a prevalent tumor condition that is correlated with metastatic spread and therapeutic resistance. Hypoxia-inducible factor 1alpha (HIF-1alpha) is a transcription factor upregulated by normal cells in hypoxia. HIF-1alpha binds the hypoxia-responsive element (HRE) in the promoter region of adaptive genes such as VEGF and erythropoietin, to stimulate gene transcription. We sought to determine if hypoxic cancers upregulate HIF-1alpha and whether hypoxia can be used as a gene therapy target. Methods: Human cancer cell lines AsPC1 (pancreatic), PLC5 (hepatoma), HCT8 (colorectal), and RF1 (gastric) and the murine cancer cell line CT26 (colorectal) were incubated in hypoxia (1%O2) and normoxia (21%O2) for HIF-1alpha ELISA and western blots. A hypoxia-responsive promoter was constructed by multimerizing the HRE from VEGF (10xHRE), and cloned into a luciferase vector for functional assay. CT26 liver metastases in syngeneic Balb/C mice (n = 8) were stained immunohistochemically for HIF-1alpha. Results: ELISA demonstrated greater HIF-1alpha levels in hypoxia versus normoxia in all cell lines (p < 0.01): CT26 (6.7-fold), AsPC1 (7.3-fold), PLC5 (5.4-fold), HCT8 (8.8-fold), and RF1 (5.9-fold). All tested cell lines demonstrated greater HIF-1alpha levels in hypoxia by western blot. Luciferase activity was higher in hypoxic cells transfected with the 10xHRE promoter (p < 0.01): CT26 (33-fold), AsPC1 (49-fold), PLC5 (29-fold), HCT8 (65-fold), and RF1 (30-fold). HIF-1alpha staining was confined to tumor-bearing regions of CT26 liver metastases. Conclusions: Hypoxia significantly upregulates HIF-1alpha expression in cancer cells. Hypoxia, a negative tumor characteristic, can thus be employed to target gene expression for cancer gene therapy.