Using oligonucleotide expression microarrays we have examined the modulation of gene expression in the DU145 prostate cancer cell line. Our findings confirm that the Egr1 transcription factor is rapidly and transiently upregulated by hypoxia. Furthermore, we have demonstrated that HIF-1alpha mRNA is also transiently upregulated, as is its target gene VEGF. To elucidate the mechanism of the transcriptional upregulation of the HIF-1alpha gene, we have shown that Egr1 is able to directly bind to the HIF-1alpha promoter using chromatin immunoprecipitation. We also provide evidence that the binding of Egr1 is necessary for the trans-activation of the HIF-1alpha promoter. These studies highlight the importance for the Egr1 transcription factor in the hypoxic response in cultured prostate cancer cell lines, and indicate that the response of Egr1 is upstream of HIF-1 in these cells. These studies are the first demonstration that the HIF-1alpha transcription factor is targeted directly by Egr1 in hypoxia.