Two members of the matrix metalloproteinase (MMP)1 family of enzymes are expressed at elevated levels in highly aggressive human tumor cells and have been implicated in the catalytic functions of extracellular proteolysis. The zymogen forms of these enzymes are designated proMMP-2 and proMMP-9, also known as 72kDa and 92kDa type IV collagenases/gelatinases, respectively. The MMP family of enzymes can be activated in vitro by a number of compounds including the organomercurial 4-aminophenylmercuric acetate (APMA). The natural or in vivo activators of MMP-2 and MMP-9 are at present unknown. A partially purified preparation of MMP-9 was used to immunize mice for the isolation of monoclonal antibodies (mAbs). Three IgG1 mAbs were identified by immunoreactivity with purified MMP-9 and are designated 6-6B, 7-11C, and 8-3H. These mAbs react specifically with MMP-9 by ELISA and Western blot. Additionally, these mAbs react with N-glycanase treated 92kDa protein. These mAbs were tested for their ability to inhibit enzyme activation in a radio-labeled gelatin assay. The 6-6B mAb inhibited the activation of MMP-9, but had no effect on MMP-2. These mAbs are highly specific to human MMP-9 and the 6-6B mAb will be extremely useful for examining the autolytic and catalytic activity of MMP-9 in normal and abnormal biological processes.