During certain developmental processes, as well as during tumor progression, polarized epithelial cells integrated within multicellular structures convert into scattered, freely migrating fibroblast-like cells. Despite the biological and clinical importance of this phenomenon, the intracellular biochemical cascades that control the switch between the epithelial and mesenchymal phenotypes have not been elucidated. Using Madin-Darby canine kidney (MDCK) cells (clone C7) as a model system, we have assessed the potential role of the mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) cascade in the modulation of epithelial plasticity. When grown in three-dimensional collagen gels, MDCK-C7 cells form spherical cysts composed of polarized epithelial cells circumscribing a central lumen. This morphogenetic behavior is profoundly subverted in MDCK-C7 cells expressing a constitutively active MAPK/ERK kinase 1 (caMEK1) mutant (C7-caMEK1 cells). When suspended in collagen gels, C7-caMEK1 cells assume an elongated fibroblastoid shape and are unable to generate multicellular cysts. In addition, when seeded onto the surface of a collagen gel, C7-caMEK1 cells penetrate extensively into the underlying matrix, unlike wild-type and mock-transfected MDCK-C7 cells, which remain confined to the surface of the gel. Similar changes in morphogenetic and invasive properties are observed in MDCK-C7F cells, a nontransfected, stably dedifferentiated derivative of MDCK-C7 cells that expresses substantially increased ERK2 activity. Both C7-caMEK1 and MDCK-C7F cells but not wild-type or mock-transfected MDCK-C7 cells express activated M(r) 72,000 gelatinase A [matrix metalloproteinase (MMP)-2] as well as elevated levels of membrane type-1 MMP. Synthetic MMP inhibitors as well as recombinant tissue inhibitor of metalloproteinases 2 and 3 suppress the invasion of collagen gels and restore the capacity of C7-caMEK1 cells to form cysts, thereby implicating the membrane type-1 MMP/MMP-2 proteolytic system in epithelial cell invasiveness and loss of multicellular organization. Taken together, our data demonstrate that increased activity of the MEK1-ERK2 signaling module in MDCK-C7 cells is associated with failure of morphogenesis and expression of a highly invasive phenotype. Sustained activation of the MAPK cascade therefore results in the destabilization of the three-dimensional architecture and the conversion of polarized epithelial cells into migrating mesenchymal-like cells.