Hypoxia-inducible factor 1 (HIF-1) is a key regulator of gene expression in response to hypoxia. This study demonstrates that HIF-1 is activated in various mammalian cell lines, including those that do not express the erythropoietin (EPO) gene. HIF-1 binds to a specific DNA sequence in the EPO gene enhancer, which is required for hypoxic activation of transcription. The composition of the HIF-1 DNA binding complex and its subunit appear to be similar in both EPO-producing and non-EPO-producing cells. Transcription of reporter genes containing the EPO gene enhancer is induced by hypoxia in non-EPO-producing cells, and mutations that eliminate HIF-1 binding eliminate inducibility. These results suggest that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia. Cells sense and respond to extracellular and intracellular stimuli to maintain homeostasis, and hypoxia is one of the most fundamental of all environmental stimuli. A major physiologic mechanism by which mammals respond to tissue hypoxia is through stimulation of erythropoiesis, resulting in an increased blood oxygen-carrying capacity. Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. EPO RNA levels increase several hundredfold in rodent liver and kidney in response to hypoxia or anemia. Hypoxia also induces EPO RNA transcription in Hep3B human hepatoblastoma cells, demonstrating that the same cell type can sense hypoxia and respond by increasing EPO RNA levels. The production of EPO and subsequent increase in erythropoiesis thus provide a major homeostatic mechanism for maintaining tissue oxygenation. A major research objective in our laboratory has been to identify the cis-acting DNA sequences and trans-acting protein factors that regulate hypoxia-inducible human EPO gene expression. Liver-specific DNase I-hypersensitive sites have been identified in the human EPO gene 3'-flanking sequence. In addition, a 256-nt fragment that encompasses the hypersensitive sites functioned as a hypoxia-inducible enhancer when cloned 3' to a simian virus 40 (SV40) promoter–chloramphenicol acetyltransferase (CAT) reporter gene and transiently expressed in Hep3B cells. Deletion and scanning-mutagenesis studies further delimited the enhancer to a 50-nt sequence that was functionally tripartite: site 1 (nt 4–12) and site 2 (nt 19–23) were absolutely required for hypoxic induction, whereas site 3 (nt 26–48), a putative thyroid/steroid hormone receptor binding siteHypoxia-inducible factor 1 (HIF-1) is a key regulator of gene expression in response to hypoxia. This study demonstrates that HIF-1 is activated in various mammalian cell lines, including those that do not express the erythropoietin (EPO) gene. HIF-1 binds to a specific DNA sequence in the EPO gene enhancer, which is required for hypoxic activation of transcription. The composition of the HIF-1 DNA binding complex and its subunit appear to be similar in both EPO-producing and non-EPO-producing cells. Transcription of reporter genes containing the EPO gene enhancer is induced by hypoxia in non-EPO-producing cells, and mutations that eliminate HIF-1 binding eliminate inducibility. These results suggest that HIF-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia. Cells sense and respond to extracellular and intracellular stimuli to maintain homeostasis, and hypoxia is one of the most fundamental of all environmental stimuli. A major physiologic mechanism by which mammals respond to tissue hypoxia is through stimulation of erythropoiesis, resulting in an increased blood oxygen-carrying capacity. Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. EPO RNA levels increase several hundredfold in rodent liver and kidney in response to hypoxia or anemia. Hypoxia also induces EPO RNA transcription in Hep3B human hepatoblastoma cells, demonstrating that the same cell type can sense hypoxia and respond by increasing EPO RNA levels. The production of EPO and subsequent increase in erythropoiesis thus provide a major homeostatic mechanism for maintaining tissue oxygenation. A major research objective in our laboratory has been to identify the cis-acting DNA sequences and trans-acting protein factors that regulate hypoxia-inducible human EPO gene expression. Liver-specific DNase I-hypersensitive sites have been identified in the human EPO gene 3'-flanking sequence. In addition, a 256-nt fragment that encompasses the hypersensitive sites functioned as a hypoxia-inducible enhancer when cloned 3' to a simian virus 40 (SV40) promoter–chloramphenicol acetyltransferase (CAT) reporter gene and transiently expressed in Hep3B cells. Deletion and scanning-mutagenesis studies further delimited the enhancer to a 50-nt sequence that was functionally tripartite: site 1 (nt 4–12) and site 2 (nt 19–23) were absolutely required for hypoxic induction, whereas site 3 (nt 26–48), a putative thyroid/steroid hormone receptor binding site