To examine MMP-9 activation in a cellular setting we employed cultures of human tumor cells that were induced to produce MMP-9 over a 200-fold concentration range (0.03 to 8.1 nM). The secreted levels of TIMPs in all the induced cultures remain relatively constant at 1-4 nM. Quantitation of the zymogen/active enzyme status of MMP-9 in the cultures indicates that even in the presence of potential activators, the molar ratio of endogenous MMP-9 to TIMP dictates whether proMMP-9 activation can progress. When the MMP-9/TIMP ratio exceeds 1.0, MMP-9 activation progresses, but only via an interacting protease cascade involving plasmin and stromelysin 1 (MMP-3). Plasmin, generated by the endogenous plasminogen activator (uPA), is not an efficient activator of proMMP-9. Plasmin, however, is very efficient at generating active MMP-3 from exogenously added proMMP-3. The activated MMP-3, when its concentration exceeds that to TIMP, becomes a potent activator of proMMP-9. Addition to the cultures of already-activated MMP-3 relinquishes the requirement for plasminogen and proMMP-3 additions and results in direct activation of the endogenous proMMP-9. The activated MMP-9 enhances the invasive phenotype of the cultured cells as their ability to transverse basement membrane is significantly increased following zymogen activation. That this enhanced tissue remodeling capability is due to the activation of MMP-9 is demonstrated through the use of a specific anti-MMP-9-blocking monoclonal antibody.