Matrix metalloproteinase 9 (MMP-9), a member of MMP family, is involved in many physiological processes, including cardiovascular disease (CVD). Tumor necrosis factor-alpha (TNF-alpha) is considered a cytokine with pleiotropic biological capabilities and leads to the process of CVD when TNF-alpha is abnormally released and stimulates MMP-9 expression and activation. In this study, we investigated the molecular mechanism of TNF-alpha-regulated MMP-9 expression. The experimental results confirm that TNF-alpha could upregulate MMP-9 expression in heart myoblast H9c2 cells of rat. To evaluate the MMP-9 regulation at transcriptional level, a DNA fragment of 2.2 kb (-2168/+18) of human mmp-9 promoter region was cloned and constructed in a vector of luciferase reporter gene. The 2.2-kb sequences were identified as having three candidate nuclear factor-kappa B (NF-kappa B) binding sites: NF-kappa B I (-1418/-1409), NF-kappa B II (-626/-617), and NF-kappa B III (-353/-345). A series of reporter vectors with the mutated NF-kappa B sites of mmp-9 promoter sequences were constructed and transfected into H9c2 cells. The results show that the NF-kappa B II binding site (-626/-617) within the promoter region of mmp-9 plays a key role in upregulation of mmp-9 expression by TNF-alpha induction. In addition, we also first identified that the NF-kappa B I, similar to c-Rel, might be one of the NF-kappa B families to regulate mmp-9 expression.