Keratinocyte migration is an absolute requirement for correct epithelialization during the process of wound healing. This process requires changes in extracellular matrix ligand expression as well as changes in ligand-binding affinity of the corresponding cellular integrins. In this study, we attempt to understand the role of laminin 5 in migration by investigating the integrin-mediated interactions of migrating keratinocytes with their newly synthesized laminin 5. We chose to induce migration of freshly isolated NHK in vitro by exposing them to TGF-β1 which, in addition to promoting epithelial cell migration, is also known to prevent cell proliferation. This important feature allowed the study to be focused on cell migration without interfering with cell proliferation. We confirm that keratinocyte migration on plastic, fibronectin or collagen IV substrates requires endogenous laminin 5 deposition, which is predominantly detected under its unprocessed form. Despite a crucial role for laminin 5 in migration, we show that this process is accompanied by a significant decrease in adhesion to purified laminin 5. Moreover, we provide evidence that the α3β1 integrin interaction with newly synthesized laminin 5 renders the cells more adherent and retards migration. Conversely, we provide evidence that the α2β1 integrin-laminin 5 interaction is absolutely required for keratinocyte migration and that the α2β1 integrin is responsible for cell spreading on laminin 5. Finally, we demonstrate that the α2β1 integrin binding to laminin 5 occurs within the short arm of the γ2 subunit.