Hypoxia-inducible factor 1 (HIF-1) is a DNA binding activity detected in nuclear extracts from Hep3B cells cultured in 1% O2 but not in extracts from cells cultured in 20% O2. HIF-1 binds to a sequence within the human erythropoietin gene enhancer that is required for hypoxic activation of transcription. Induction of HIF-1 is inhibited by cycloheximide, which also inhibits induction of erythropoietin RNA. We now demonstrate that induction of both HIF-1 and erythropoietin RNA is inhibited by the protein kinase inhibitor 2-aminopurine. HIF-1 binding to DNA was eliminated by phosphatase treatment of nuclear extracts. Actinomycin D also inhibited HIF-1 induction, suggesting that de novo transcription is required. The kinetics of HIF-1 induction by hypoxia paralleled the kinetics of erythropoietin gene transcriptional induction. HIF-1 DNA binding activity decayed rapidly when hypoxic cells were exposed to increased oxygen tension. In vitro, the kinetics of HIF-1 association with, and dissociation from, its binding site were extremely rapid, with a t1/2 for both processes of < 1 min. These findings are consistent with the proposed function of HIF-1 as a physiologic regulator of gene expression that responds to changes in cellular oxygen tension. Methylation interference analysis indicated that HIF-1 makes specific contacts with DNA in the major groove.