Hypoxia-induced up-regulation of vascular endothelial growth factor (VEGF) expression is a critical event leading to tumor neovascularization. Hypoxia stimulates hypoxia-inducible factor-1α (HIF-1α), a transcriptional activator of VEGF. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, is also induced by hypoxia. We reported previously that COX-2 inhibition prevents hypoxic up-regulation of VEGF in human prostate cancer cells and that prostaglandin E2 (PGE2) restores hypoxic effects on VEGF. We hypothesized that PGE2 mediates hypoxic effects on VEGF by modulating HIF-1α expression. Addition of PGE2 to PC-3ML human prostate cancer cells had no effect on HIF-1α mRNA levels. However, PGE2 significantly increased HIF-1α protein levels, particularly in the nucleus. This effect of PGE2 largely results from the promotion of HIF-1α translocation from the cytosol to the nucleus. PGE2 addition to PC-3 ML cells transfected with a GFP-HIF-1α vector induced a time-dependent nuclear accumulation of the HIF-1α protein. Two selective COX-2 inhibitors, meloxicam and NS398, decreased HIF-1α levels and nuclear localization, under both normoxic and hypoxic conditions. Of several prostaglandins tested, only PGE2 reversed the effects of a COX-2 inhibitor in hypoxic cells. Finally, PGE2 effects on HIF-1α were specifically inhibited by PD98059 (a MAPK inhibitor). These data demonstrate that PGE2 production via COX-2-catalyzed pathway plays a critical role in HIF-1α regulation by hypoxia and imply that COX-2 inhibitors can prevent hypoxic induction of HIF-mediated gene transcription in cancer cells.