Matrix metalloproteinases (MMPs) and integrins have been implicated in a variety of processes involved in tumor progression. To evaluate the individual roles of integrin alphavbeta3 and membrane-type 1 matrix metalloproteinase (MT1-MMP), as well as the effects of their joint expression on tumor cell functions, MCF7 breast carcinoma cells were transfected stably with either the MT1-MMP, the beta3 integrin subunit or both MT1-MMP and beta3 cDNAs. MT1-MMP expression is accompanied by the functional activation of integrin alphaVbeta3, thereby increasing vitronectin-mediated adhesion and migration of MCF7 cells transfected with MT1-MMP and integrin alphaVbeta3. MT1-MMP-dependent functional activation of alphaVbeta3 correlates with modification(s) of the beta3 subunit, including its higher electrophoretic mobility and affected the LM609-binding site. MCF7 cells jointly expressing MT1-MMP and alphaVbeta3 were the most efficient in adhesion to the recombinant C-terminal domain of MMP-2 as well as in generating soluble and cell surface associated mature MMP-2 enzyme. These findings suggest a mechanism of selective docking of MMP-2 at tumor cell surfaces, specifically at the sites that include MT1-MMP and activated integrin alphaVbeta3. These mechanisms may provide a link between spatial regulation of focal proteolysis by the cell surface associated MMPs and the regulation of integrin-mediated motility of tumor cells.