This paper investigates the characterization and regulation of Hypoxia-inducible Factor 1 (HIF-1) in response to hypoxia. HIF-1 is a DNA binding activity detected in nuclear extracts from Hep3B cells cultured under low oxygen conditions (1% O2) but not in extracts from cells cultured under high oxygen conditions (20% O2). HIF-1 binds to a specific sequence within the human erythropoietin (EPO) gene enhancer, which is essential for hypoxic activation of transcription. The induction of HIF-1 is inhibited by cycloheximide and 2-aminopurine, suggesting that de novo protein synthesis and protein kinase activity are required for HIF-1 induction. Actinomycin D also inhibits HIF-1 induction, indicating that de novo transcription is necessary. The kinetics of HIF-1 induction by hypoxia are similar to those of EPO gene transcriptional induction. HIF-1 binding activity decays rapidly when hypoxic cells are exposed to increased oxygen tension. In vitro studies show that HIF-1 binding to DNA is rapid, with a half-life of less than 1 minute. Methylation interference analysis reveals that HIF-1 makes specific contacts with DNA in the major groove. These findings support the role of HIF-1 as a physiological regulator of gene expression that responds to changes in cellular oxygen tension.This paper investigates the characterization and regulation of Hypoxia-inducible Factor 1 (HIF-1) in response to hypoxia. HIF-1 is a DNA binding activity detected in nuclear extracts from Hep3B cells cultured under low oxygen conditions (1% O2) but not in extracts from cells cultured under high oxygen conditions (20% O2). HIF-1 binds to a specific sequence within the human erythropoietin (EPO) gene enhancer, which is essential for hypoxic activation of transcription. The induction of HIF-1 is inhibited by cycloheximide and 2-aminopurine, suggesting that de novo protein synthesis and protein kinase activity are required for HIF-1 induction. Actinomycin D also inhibits HIF-1 induction, indicating that de novo transcription is necessary. The kinetics of HIF-1 induction by hypoxia are similar to those of EPO gene transcriptional induction. HIF-1 binding activity decays rapidly when hypoxic cells are exposed to increased oxygen tension. In vitro studies show that HIF-1 binding to DNA is rapid, with a half-life of less than 1 minute. Methylation interference analysis reveals that HIF-1 makes specific contacts with DNA in the major groove. These findings support the role of HIF-1 as a physiological regulator of gene expression that responds to changes in cellular oxygen tension.