The integrin alpha6beta4 has been implicated in two apparently contrasting processes, i.e., the formation of stable adhesions, and cell migration and invasion. To study the dynamic properties of alpha6beta4 in live cells two different beta4-chimeras were stably expressed in beta4-deficient PA-JEB keratinocytes. One chimera consisted of full-length beta4 fused to EGFP at its carboxy terminus (beta4-EGFP). In a second chimera the extracellular part of beta4 was replaced by EGFP (EGFP-beta4), thereby rendering it incapable of associating with alpha6 and thus of binding to laminin-5. Both chimeras induce the formation of hemidesmosome-like structures, which contain plectin and often also BP180 and BP230. During cell migration and division, the beta4-EGFP and EGFP-beta4 hemidesmosomes disappear, and a proportion of the beta4-EGFP, but not of the EGFP-beta4 molecules, become part of retraction fibers, which are occasionally ripped from the cell membrane, thereby leaving "footprints" of the migrating cell. PA-JEB cells expressing beta4-EGFP migrate considerably more slowly than those that express EGFP-beta4. Studies with a beta4-EGFP mutant that is unable to interact with plectin and thus with the cytoskeleton (beta4(R1281W)-EGFP) suggest that the stabilization of the interaction between alpha6beta4 and LN-5, rather than the increased adhesion to LN-5, is responsible for the inhibition of migration. Consistent with this, photobleaching and recovery experiments revealed that the interaction of beta4 with plectin renders the bond between alpha6beta4 and laminin-5 more stable, i.e., beta4-EGFP is less dynamic than beta4(R1281W)-EGFP. On the other hand, when alpha6beta4 is bound to laminin-5, the binding dynamics of beta4 to plectin are increased, i.e., beta4-EGFP is more dynamic than EGFP-beta4. We suggest that the stability of the interaction between alpha6beta4 and laminin-5 is influenced by the clustering of alpha6beta4 through the deposition of laminin-5 underneath the cells. This clustering ultimately determines whether alpha6beta4 will inhibit cell migration or not.