The biological functions of matrix metalloproteinases (MMPs) extend beyond extracellular matrix degradation. Non-proteolytic activities of MMPs are just beginning to become understood. The role of proMMPs in cell migration was herein evaluated using transfected COS-1 cells with various proMMP cDNAs employed in a Transwell chamber migration assay. Latent MMP-2 and MMP-9 enhanced cell migration to a greater extent than latent MMP-1,. 3,. 11 and. 28. To examine if proteolytic activity is required for MMP-enhanced cell migration, three experimental approaches including a fluorogenic substrate degradation assay, transfection of cells with catalytically inactive mutant of MMP cDNAs, and addition of MMP inhibitors were utilized. The mechanism underlying the non-proteolytic enhancement of cell migration by MMPs was evaluated by the structure-function relationship of MMP-9 on cell motility. A domain swapping approach was utilized to demonstrate the role of the hemopexin (PEX) domain of proMMP-9 in cell migration when examined by a Transwell chamber assay and by a phagokinetic assay. TIMP-1, which interacts with the PEX domain of proMMP-9, inhibited cell migration whereas TIMP-2 had no effect. Furthermore, using a biochemical approach, it was demonstrated that dimerization of MMP-9 through the PEX domain appears necessary for MMP-9-enhanced cell migration. Following a series of substitution mutations within the MMP-9 PEX domain, blade IV was shown to be critical for homodimerization, whereas blade I was required for heterodimerization with CD44. Both blade I and IV mutants showed diminished enhancement of cell migration compared to wild type MMP-9 transfected cells. Peptides mimicking motifs of the outermost strands of the first and fourth blades of the MMP-9 PEX domain were designed; these peptides efficiently blocked MMP-9 dimerization and inhibited motility of COS-1 cells overexpressing MMP-9, HT-1080 and MDA-MB-435 cells. Using a shRNA approach, CD44 was found to be a critical molecule in MMP-9-mediated cell migration. An axis involving an MMP-9-CD44-EGFR signaling pathway in cell migration was identified using an antibody array and specific receptor tyrosine kinase inhibitors. In conclusion, the mechanism by which proMMP-9 can enhance cell migration was dissected. Biochemical studies led to the development of structure-based inhibitory peptides, and small molecules targeting MMP-9-mediated cell migration.