Expression of the transferrin receptor, which mediates iron uptake from transferrin, is negatively regulated post-transcriptionally by intracellular iron through iron-responsive elements in the 3'-untranslated region of the transferrin receptor mRNA. Transcriptional mechanisms are also involved in receptor expression, but these are poorly understood. In this study we have characterized the transferrin receptor promoter region and identified a functional hypoxia response element that contains a binding site for hypoxia-inducible factor-1 (HIF-1). Exposure of K562 and HeLa cells to hypoxia for 16 h resulted in a 2- to 3-fold increase in transferrin receptor mRNA expression. A motif with multipartite organization similar to the hypoxia response element of a number of hypoxia-inducible genes such as erythropoietin was identified within a 100-base pair sequence upstream of the transcriptional start site. Mutation of a site similar to the consensus HIF-binding site (HBS) in this motif attenuated the hypoxic response by 80%. Transient co-expression of the two HIF-1 subunits (HIF-1alpha and HIF-1beta) enhanced the wild type transferrin receptor promoter activity, but that which contained a mutated HBS yielded no such response. Electrophoretic mobility shift assays revealed that HIF-1 was stimulated and bound to the transferrin receptor HBS upon hypoxic challenge. Our results indicate that the transferrin receptor is a target gene for HIF-1.