To develop a potent hypoxia-inducible vector, we evaluated in the minimal SV40 promoter setting the usefulness of chimeric combinations of the early growth response factor-1 (Egr-1)-binding site (EBS) from the Egr-1 promoter, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 (PGK1) gene. In transient transfection assays, a construct (H-pGL3) containing three copies of HRE (3��HRE) gave a hypoxic induction ratio of 20.2, whereas the EBS- and MRE-containing vectors displayed no apparent induction. Interestingly, the combination of 3��HRE with either EBS or MRE resulted in a significant increase in hypoxia-responsiveness. With three-enhancer combination such as the EBS-MRE-3��HRE (E-M-H), a hypoxic induction ratio of 69 was achieved. The expression induced from E-M-H-pGL3 was 2.4-fold higher (three-fold higher by induction ratio) than that induced from H-pGL3 and even surpassed the expression from an HCMV-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs, such as that of basic fibroblast growth factor. E-M-H-pGL3 was also tested for inducibility by hypoxia mimetics and other stresses such as heat, low glucose, low pH, and hydrogen peroxide. Co2+, deferoxamine, and Ni2+ turned out to be potent inducers whereas hydrogen peroxide and low pH (6.0) worked as moderate inducers. Heat and low glucose had little effect on the inducibility. Analysis of HIF-1慣, MTF-1, and Egr-1 level in the nuclear extract indicated that their binding to corresponding DNA motifs increased in response to hypoxia. In addition, overexpression studies of HIF-1慣, MTF-1, and/or Egr-1 suggested that MTF-1 and Egr-1 synergize with HIF-1慣 for the high inducibility of E-M-H chimeric enhancer. Next, we tested the efficacy of E-M-H in vivo in a murine model of hindlimb ischemia. The expressed luciferase reporter in the ischemic calf muscle was 4-fold higher in E-M-H group than in H or HCMV group. With its high induction capacity and versatile means of modulation, the novel chimeric enhancer should find wide application in the treatment of ischemic diseases and cancer.