AbstractMembrane type‐1 matrix metalloproteinase (MT1‐MMP) and αvβ3 integrin have been directly implicated in tumor cell dissemination and metastasis. We have demonstrated that in the case of breast carcinoma MCF7 cells co‐expressing MT1‐MMP and αvβ3 integrin, the proteinase processes the pro‐αv integrin subunit, thus facilitating αvβ3 integrin maturation and cell migration on vitronectin. Our findings show that cell surface MT1‐MMP is a short‐lived protein with a life span in the range of several hours. In contrast, turnover of αvβ3 integrin is much slower. The half‐life of αvβ3 heterodimer is about 24 hr. This large difference in life span allowed us to distinguish between the effects of MT1‐MMP on cell migration brought by matrix proteolysis from those imposed through αvβ3 integrin maturation. We then modulated the enzyme's activity by a potent hydroxamate MMP inhibitor, Prinomastat (AG3340), to analyze the divergent effects of MT1‐MMP on cell migration. Although Prinomastat immediately blocked MT1‐MMP‐mediated matrix degradation, the pool of MT1‐MMP‐modified αvβ3 integrin molecules was still capable of mediating cell‐matrix interactions. To our considerable surprise, inhibition of MT1‐MMP‐dependent vitronectin proteolysis by Prinomastat allowed a several‐fold increase in migration of MCF7 cells co‐expressing MT1‐MMP and αvβ3 integrin. In contrast, long‐term Prinomastat inhibition of MT1‐MMP‐dependent pro‐αv cleavage and thus αvβ3 integrin maturation strongly inhibited cell motility. Our studies suggest that MT1‐MMP could actually promote cell migration via modification of the cell surface receptors, including αvβ3 integrin, rather than facilitate cell migration through direct cleavage of the matrix proteins. © 2003 Wiley‐Liss, Inc.