Studies of gene regulation by oxygen have recently defined the existence of a widely operative system that responds to hypoxia but not mitochondrial inhibitors and involves the induction of a DNA-binding complex termed hypoxia-inducible factor 1. This system has been implicated in the regulation of erythropoietin, certain angiogenic growth factors, and particular glycolytic isoenzymes. The glucose transporter Glut-1 is induced by both hypoxia and mitochondrial inhibitors, implying the operation of a different mechanism of oxygen sensing. To explore that possibility, we analyzed the cisacting sequences that convey these responses. An enhancer lying 5' to the mouse Glut-1 gene was found to convey responses both to hypoxia and to the mitochondrial inhibitors, azide and rotenone. However, detailed analysis of this enhancer demonstrated that distinct elements responded to hypoxia and the mitochondrial inhibitors. The response to hypoxia was mediated by sequences that contained a functionally critical, although atypical, hypoxia-inducible factor 1 binding site, whereas sequences lying approximately 100 nucleotides 5' to this site, which contained a critical serum response element, conveyed responses to the mitochondrial inhibitors. Thus, rather than reflecting an entirely different mechanism of oxygen sensing, regulation of Glut-1 gene expression by hypoxia and mitochondrial inhibitors arises from the function of two different sensing systems. One of these responds to hypoxia alone and resembles that involved in erythropoietin regulation, while the other responds to mitochondrial inhibitors and involves activation of a serum response element.