Pro-MMP2 activation is a two-step process resulting in (1) an intermediate 64 kDa form generated by the MT1-MMP activity, and (2) a mature 62 kDa form. Addition of plasminogen to HT1080 cells cultured under various conditions, or to their membrane preparation, induced a complete conversion of the intermediate MMP-2 form to the mature one, and processing of pro-MMP-9. The pro-MMP-2 activation was inhibited by plasmin inhibitors and anti-uPA antibody. These results provide evidence for involvement of the PA/plasmin system in the second step of MMP-2 activation.