Epithelial cell migration is a crucial event in wound healing, yet little is known about mechanisms whereby integrins regulate epithelial cell polarization and migration. In the present work, we demonstrate the importance of adhesion through the α3β1 integrin in promoting the stabilization of leading lamellipodia in migrating keratinocytes. We demonstrate that this integrin is found at the leading edge of migrating keratinocytes and that inhibition of α3β1 binding to laminin-5 prevents the formation of stable leading lamellipodia. Consistent with this observation, keratinocytes derived from α3β1-deficient mice fail to form stable leading lamellipodia but retain the ability to form actin-containing protrusions that rapidly extend and retract from the cell membrane. Formation of a leading lamellipodium also requires α3β1-dependent activation of Rac1, because α3β1-deficient keratinocytes show decreased activation of Rac1 compared with α3β1-expressing cells, and formation of stable leading lamellipodia can be inhibited in the latter cells by expression of the dominant negative Rac1 mutant Rac1N17. Furthermore, α3β1-deficient keratinocytes expressing constitutively active Rac1L61 failed to form stable lamellipodia when plated onto laminin-5, demonstrating that α3β1 is required for Rac1-mediated formation of a stable lamellipodium. These observations identify a crucial role for integrin-mediated adhesion and signaling in the formation of large, polarized, stable lamellipodia by migrating epithelial cells. To our knowledge, this study is the first to demonstrate that signal transduction through a specific integrin is required to direct the development of a lamellipodium from an initial protrusion and promote persistent epithelial cell migration.