The cellular response to hypoxia is mediated by the hypoxia- inducible factor-1alpha (HIF-1alpha). Although it is known that hypoxia regulates HIF-1alpha via protein stability, several in vivo studies implicated that hypoxia also increases HIF-1alpha mRNA levels as for example in pulmonary hypertension (PH). We tested whether hypoxia regulates HIF-1alpha mRNA in pulmonary artery smooth muscle cells. Hypoxia rapidly elevated HIF-1alpha mRNA levels. Actinomycin D abrogated this response. Using deletion constructs of the HIF-1alpha promoter we identified a hypoxia-sensitive promoter region harboring a putative NFkappaB consensus site. Hypoxia activated NFkappaB. Dominant-negative IkappaB diminished hypoxic induction of HIF-1alpha promoter, mRNA and protein. Chromatin immunoprecipitation, gel shift analyses and site-directed mutagenesis confirmed hypoxia-stimulated NFkappaB binding to the HIF-1alpha promoter. Finally, the PI3 kinase (PI3K) inhibitor wortmannin inhibited the activation of NFkappaB and HIF-1alpha promoter by hypoxia. These findings show that under hypoxia HIF-1alpha is a transcriptional target of NFkappaB which is activated via a PI3K-dependent pathway. This novel mechanism may explain increased levels of HIF-1alpha mRNA in PH and other disorders associated with chronic hypoxia.