Hypoxia, a hallmark of many solid tumors, is associated with angiogenesis and tumor progression. It activates a signal cascade that culminates in the stabilization of hypoxia-inducible factor-1 (HIF-1) transcription factor and activation of genes that possess hypoxia response elements. The loss of tumor suppressors such as p53 has been shown to stabilize HIF-1alpha, which is overexpressed in the majority of human cancers, and its over-expression correlates with poor prognosis and treatment failure. Here we constructed hypoxia-inducible promoters and examined their activities in murine and human cancer cells with variable p53 status. Loss of p53 function in cancer cells resulted in increased HIF-1-dependent transcriptional activity. To investigate the feasibility of exploiting the hypoxic tumor microenvironment for targeted gene therapy of cancer, we constructed retroviral vectors harboring luciferase or Escherichia coli cytosine deaminase (CD) genes under the control of the hypoxia-inducible promoter. Murine Lewis lung carcinoma (LL2) cells carrying defective p53, when retrovirally transduced with the hypoxia-inducible promoter-driven luciferase gene under hypoxic conditions, increased luciferase reporter gene expression in vitro and in vivo. Significant antitumor effects were achieved in mice bearing LL2 tumors that expressed CD driven by a hypoxia-inducible promoter after treatment with 5-fluorocytosine. These results suggest the potential applications of suicide genes, such as the CD gene, under the control of hypoxia-inducible promoters for cancer gene therapy, which may target efficiently to hypoxic regions of tumors with p53 mutations.