Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes, including the human erythropoietin (EPO) gene. This study identifies structural features of the HIF-1α subunit required for heterodimerization, DNA binding, and transactivation. HIF-1α and HIF-1β (ARNT) coimmunoprecipitated from nuclear extracts, indicating heterodimerization in the absence of DNA. In vitro-translated HIF-1α and HIF-1β formed a HIF-1/DNA complex with similar electrophoretic mobility and sequence specificity as HIF-1 in hypoxic cells. Amino acids 1–166 mediated heterodimerization with HIF-1β, while amino acids 1–390 were required for optimal DNA binding. A deletion involving the basic domain of HIF-1α eliminated DNA binding without affecting heterodimerization. Cotransfection assays showed that recombinant HIF-1α and HIF-1β activated transcription of reporter genes containing EPO enhancer sequences with intact, but not mutant, HIF-1 binding sites. Deletion of the carboxy terminus of HIF-1α markedly decreased transcriptional activation. Overexpression of a HIF-1α construct with deletions of the basic domain and carboxy terminus blocked reporter gene activation by endogenous HIF-1 in hypoxic cells. HIF-1 plays a key role in EPO gene transcriptional activation in hypoxic cells. A 3-bp substitution at site 1 eliminated enhancer activity and binding of HIF-1. Exposure of cells to 1% O₂, cobalt chloride, or desferrioxamine induced both EPO expression and HIF-1 activity with similar kinetics. Treatment of hypoxic cells with protein kinase or protein synthesis inhibitors blocked induction of EPO RNA and HIF-1 activity. In various non-EPO-producing lines, HIF-1 was induced by hypoxia and EPO 3'-flanking sequences functioned as hypoxia-inducible enhancers. Expression of genes encoding vascular endothelial growth factor and glycolytic enzymes was induced by exposure to 1% O₂, cobalt chloride, or desferrioxamine, and these genes contained HIF-1 binding sites within sequences mediating transcriptional activation in hypoxic cells. These results indicate a general role for HIF-1 in O₂ homeostasis. Purification of HIF-1 by DNA affinity chromatography and characterization of amino acid and cDNA sequences revealed that HIF-1 was a heterodimeric transcription factor of the bHLH-PAS family. The bHLH domain mediatesHypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes, including the human erythropoietin (EPO) gene. This study identifies structural features of the HIF-1α subunit required for heterodimerization, DNA binding, and transactivation. HIF-1α and HIF-1β (ARNT) coimmunoprecipitated from nuclear extracts, indicating heterodimerization in the absence of DNA. In vitro-translated HIF-1α and HIF-1β formed a HIF-1/DNA complex with similar electrophoretic mobility and sequence specificity as HIF-1 in hypoxic cells. Amino acids 1–166 mediated heterodimerization with HIF-1β, while amino acids 1–390 were required for optimal DNA binding. A deletion involving the basic domain of HIF-1α eliminated DNA binding without affecting heterodimerization. Cotransfection assays showed that recombinant HIF-1α and HIF-1β activated transcription of reporter genes containing EPO enhancer sequences with intact, but not mutant, HIF-1 binding sites. Deletion of the carboxy terminus of HIF-1α markedly decreased transcriptional activation. Overexpression of a HIF-1α construct with deletions of the basic domain and carboxy terminus blocked reporter gene activation by endogenous HIF-1 in hypoxic cells. HIF-1 plays a key role in EPO gene transcriptional activation in hypoxic cells. A 3-bp substitution at site 1 eliminated enhancer activity and binding of HIF-1. Exposure of cells to 1% O₂, cobalt chloride, or desferrioxamine induced both EPO expression and HIF-1 activity with similar kinetics. Treatment of hypoxic cells with protein kinase or protein synthesis inhibitors blocked induction of EPO RNA and HIF-1 activity. In various non-EPO-producing lines, HIF-1 was induced by hypoxia and EPO 3'-flanking sequences functioned as hypoxia-inducible enhancers. Expression of genes encoding vascular endothelial growth factor and glycolytic enzymes was induced by exposure to 1% O₂, cobalt chloride, or desferrioxamine, and these genes contained HIF-1 binding sites within sequences mediating transcriptional activation in hypoxic cells. These results indicate a general role for HIF-1 in O₂ homeostasis. Purification of HIF-1 by DNA affinity chromatography and characterization of amino acid and cDNA sequences revealed that HIF-1 was a heterodimeric transcription factor of the bHLH-PAS family. The bHLH domain mediates