Erythropoietin (Epo) production in response to hypoxia or cobalt is primarily mediated by activation of transcription of the Epo gene. Recently an hypoxia responsive enhancer was identified in the 3′ flanking region of the mouse and human Epo genes. Using functional analysis in Hep 3B cells we define here the minimal enhancer element as a 29-bp segment starting at the Apa1 site in the 3′ flanking region of the human Epo gene. Mutagenesis studies of the minimal element identified three different areas that are necessary for full enhancer activity. Electrophoretic mobility shift assays show the presence of hypoxia- and/or cobalt-inducible nuclear DNA-binding proteins that bind to one of the active sites of the enhancer. Induction of hypoxia- binding activity was abolished by Anisomycin, a potent protein synthesis inhibitor, suggesting that de novo protein synthesis is necessary for the activation process. Further characterization of DNA- binding proteins by use of UV light crosslinking identified a protein of molecular weight of approximately 120-Kd that was present only in hypoxic extracts. This protein was found to be present in hypoxic nuclear extracts from both Epo-producing and non-Epo-producing cells, suggesting that it may be involved in a more generalized mechanism of cellular response to hypoxia.