Vascular endothelial growth factor (VEGF) expression is highly stimulated by hypoxia, both in vitro and in vivo. Recent findings suggest that the VEGF gene utilizes an oxygen sensing mechanism similar to the one used by the erythropoietin gene. The genomic sequences that control the VEGF response to hypoxia are, however, largely unknown. In utilizing transient transfection assays in HeLa cells we determined that hypoxia/cobalt responsive enhancer elements are present at the 5' and 3' flanking regions of the human VEGF gene. The 3' enhancer is contained in a 160 bp fragment located about 60 bp downstream of the polyadenylation site. It contains a sequence stretch of about 12 bp which are highly homologous to sequences in the erythropoietin hypoxia-responsive enhancer. The 5' flanking enhancer is contained in a 100 bp fragment located about 800 bp upstream of the start site. This fragment does not contain significant homologies with the erythropoietin enhancer. Thus, it appears that the response to hypoxia of the VEGF gene is controlled by two regulatory elements; one which may be related to the erythropoietin enhancer and a second, which appears to be a completely unrelated sequence.