Matrix metalloproteinase (MMP)-9 plays an important role in cardiovascular events. However, the mechanisms underlying in vivo activation of MMP-9 are largely unknown. We investigated the secretion and activation of MMP-9 under a cell-to-cell interaction, and the effects of hypoxia and cytokine. Human umbilical vein endothelial cell (HUVEC) and THP-1 (human monocyte cell line) were cultured individually, or cocultured under normoxic and hypoxic conditions. In a coculture of HUVEC and THP-1, proMMP-9 secretion was increased twofold compared with individual culture of HUVEC and THP-1, whereas MMP-2 secretion was unchanged. The increase in proMMP-9 secretion was suppressed by antiadhesion molecule antibodies and mitogen-activated protein kinase inhibitors, PD98059 (MAPK/ERK kinase1 inhibitor) and SP600125 (Jun N-terminal kinase inhibitor). ProMMP-9 secretion was increased by tumor necrosis factor (TNF)-α at 50 ng/ml (P < 0.05) but was not activated under normoxic (20%) conditions. ProMMP-9 in coculture was activated under hypoxic (<1%) conditions, and was potentiated by TNF-α (both P < 0.05). To further investigate the mechanism of hypoxia-induced MMP-9 activation, heat shock protein (Hsp)90, which was suggested to be related to MMP-9 activation, was measured by Western blot analysis. The ratio of Hsp90 to glyceraldehyde-3-phosphate dehydrogenase was increased in hypoxic (<1%) coculture conditions with TNF-α (P < 0.05). Treatment with geldanamycin and 17-DMAG (Hsp90 inhibitor) suppressed the active form of MMP-9. Cell-to-cell interaction between endothelial cells and monocytes promotes proMMP-9 synthesis and secretion. Hypoxia and inflammation are suggested to play an important role in activating proMMP-9, presumably via Hsp90.