The integrin alphavbeta6 is expressed on a variety of epithelial cells during dynamic processes including organogenesis, tissue injury and malignant transformation. However, because of the lack of tools to specifically inhibit the function of this integrin, little is known about its effects on cell behavior. To directly examine the role of this integrin in cell migration, we used keratinocytes derived from wild-type mice or mice expressing a null mutation in the beta6 subunit (beta6-/-) to perform migration assays in vitro. Migration on the known alphavbeta6 ligand, fibronectin was reduced in keratinocytes from beta6-/- mice. Interestingly, keratinocytes from beta6-/- mice also demonstrated markedly reduced migration on vitronectin, a protein not previously known to be a ligand for alphavbeta6. An anti-alphavbeta6 monoclonal antibody 10D5, generated by immunization of beta6-/- mice with murine keratinocytes, inhibited adhesion and migration of wild-type keratinocyte on both vitronectin and fibronectin to levels similar to those seen with keratinocytes from beta6-/- mice. alphavbeta6-mediated migration on both ligands was dramatically augmented by treatment with phorbol myrisate acetate (PMA) or with hepatocyte growth factor, and augmentation of migration by either stimulus could be abolished by the PKC inhibitor GF109203X, suggesting a critical role for PKC in enhancement of alphavbeta6-mediated cell migration.