Hypoxia initiates a variety of physiological responses and plays a crucial role in the pathogenesis of several human diseases. In this study, we attempted to develop mammalian expression vectors that respond to hypoxia by utilizing enhancer elements such as hypoxia-responsive elements (HREs), metal-responsive elements (MREs), and the Egr-1 binding site (EBS) that could confer transcriptional activation under conditions of hypoxia. The individual enhancer element was cloned into the pGL3-Promoter Vector (Promega) having minimal SV40 promoter and luciferase reporter gene and evaluated for hypoxia-responsiveness. When transient transfection assays were performed in HeLa cells, the p3XHREluc, pMREluc and pEBSluc constructs displayed 14-, 1.8- and 2-fold increased luciferase activity in hypoxia (1% O2), respectively. Next, we tested different combinations of the 3XHRE, MRE, and EBS for further enhancement of hypoxia-responsiveness. Interestingly, the two constructs, pMRE/3XHREluc and pEBS/3XHREluc, gave 26- and 16-fold increased luciferase activity, indicating additive effects and independent contributions from the two enhancers. Similar observations were made in wild type (C4.5) CHO cells. However, these hypoxia-responsiveness was not apparent in HIF-1alpha-deficient (Ka13) CHO cells when tested with the vectors having either single or two enhancers, which suggests that HIF-1alpha is an important factor in mediating hypoxia-responsiveness of the 3XHRE, MRE, EBS, and their combinations. Currently optimization of the vectors in terms of number, order, and orientation of different enhancers are underway. Our chimeric hypoxia-responsive vectors could find wide application in therapeutic gene expression for the treatment of ischemic diseases and cancer.