Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a membrane-anchored matrix metalloproteinase (MMP) that is frequently associated with processes involving tissue remodelling and cell migration. We have examined MT1-MMP expression and subcellular distribution as a function of MCF10A mammary epithelial cell migration using an in vitro outgrowth migration assay. Stronger expression of MT1-MMP was observed at the mRNA and at the protein level in cells at the periphery of the outgrowth. As shown by videomicroscopy, these cells were involved in an orientated cell migration, in contrast to stationary cells distant from the periphery. Furthermore, MT1-MMP was mainly distributed in lamellipodia of migratory cells, as well as at their basal surface in contact with the substrate. Laminin-5 (Ln-5), a recently described substrate for MT1-MMP, was deposited preferentially in the matrix by migratory cells. Fragments of the gamma2 subunit of Ln-5 were also identified in migratory cultures of MCF10A cells, attesting to its proteolytic degradation. These fragments corresponded in size to those we observed after incubation of purified human Ln-5 with the recombinant catalytic domain of human MT1-MMP. We also show that anti-Ln5 blocking antibodies, MMP inhibitors (BB94 and TIMP-2) and MT1-MMP antisense oligonucleotides significantly decreased MCF10A cell migration. Taken together, these observations demonstrate that MT1-MMP is spatially and temporally regulated during MCF10A cell migration, and suggest that MT1-MMP-mediated pericellular proteolysis of Ln-5 gamma2 chain could contribute to this process.