Matrix metalloproteinases (MMP) are central to tissue penetration by cancer cells, as tumors expand and form metastases, but the mechanism by which MMP-2 contributes to cancer cell migration is not well understood. In the present experiments, both a broad-spectrum MMP inhibitor and the isolated collagen binding domain (CBD) from MMP-2 inhibited cell migration on native type I collagen. These results verified the involvement of MMPs in general and showed that MMP-2, specifically, contributes to cell migration by a mechanism involving MMP-2 interaction with collagen. To exclude potential overlapping effects of MMP-9, additional experiments showed that MMP-2 also contributed to migration of MMP-9-/- cells. To investigate whether the homologous CBD from human fibronectin also inhibited cell migration, we first showed that fragmentation of fibronectin is a feature of breast cancer tumors and that several fragments contained the CBD. However, the recombinant fibronectin domain did not alter cell migration on collagen. This lack of effect on cell migration was explored in competitive protein-protein binding assays, which showed that the affinity of MMP-2 for collagen exceeds that of fibronectin. Furthermore, whereas the isolated MMP-2 CBD inhibited the gelatinolytic activities of MMP-2 and tumor extracts, such an inhibition was not characteristic of the corresponding fibronectin domain. Together, our results provide evidence that MMP-2 is an important determinant of cancer cell behavior but is not inhibited by the collagen binding segment of fibronectin.